Coordinated decrease and increase of gene expression of more than one gene using transgenic constructs

ABSTRACT

The present invention is directed to nucleic acid molecules and nucleic acid constructs, and other agents associated with simultaneous up- and down-regulation of expression of RNAs. Specifically it includes methods of simultaneously enhancing the expression of a first RNA at the same time as suppressing the expression of a second RNA. The present invention also specifically provides constructs capable of simultaneously enhancing the expression of a first RNA while at the same time suppressing the expression of a second RNA, methods for utilizing such agents and plants containing such agents. The present invention also provides other constructs including polycistronic constructs.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 10/393,347, filed Mar. 21, 2003, which claims priority under 35U.S.C. §19(e) to U.S. Provisional Application No. 60/390,185, filed Jun.21, 2002, each of which is herein incorporated by reference in itsentirety.

INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the sequence listing on diskette,containing the file named “16517.265.SeqList.txt”, which is 24,734 bytesin size (measured in MS-DOS), and which was created on Sep. 24, 2003,are herein incorporated by reference.

FIELD OF THE INVENTION

[0003] The present invention is directed to nucleic acid molecules andnucleic acid constructs, and other agents associated with simultaneousup- and down-regulation of expression of RNAs. Specifically it includesmethods of simultaneously enhancing the expression of a first RNA at thesame time as suppressing the expression of a second RNA using a singleconstruct. The present invention also specifically provides constructscapable of simultaneously enhancing the expression of a first RNA whileat the same time suppressing the expression of a second RNA, methods forutilizing such constructs and plants containing such constructs. Thepresent invention also provides other constructs including polycistronicconstructs.

BACKGROUND OF THE INVENTION

[0004] Many complex biochemical pathways have now been manipulatedgenetically, usually by suppression or over-expression of single genes.Further exploitation of the potential for plant genetic manipulationwill require the coordinated manipulation of multiple genes in apathway. A number of approaches have been used to combine transgenes inone plant—including sexual crossing, retransformation,co-transformation, and the use of linked transgenes. A chimerictransgene with linked partial gene sequences can be used to coordinatelysuppress numerous plant endogenous genes. Constructs modeled on viralpolyproteins can be used to simultaneously introduce multiple codinggenes into plant cells (for a review, see Halpin et al., Plant Mol.Biol. 47:295-310 (2001)).

[0005] Enhancement of gene expression in plants may occur through theintroduction of extra copies of coding sequences of the genes into aplant cell or, preferably, the incorporation of extra copies of codingsequences of the gene into the plant genome. Over-expression may alsooccur through increasing the activities of the regulatory mechanismsthat regulate the expression of genes, i.e., up-regulation of the geneexpression.

[0006] Suppression of gene expression, also known as silencing of genes,in plants occurs at both the transcriptional level andpost-transcriptional level. There are various methods for thesuppression of expression of endogenous sequences in a host cell. Suchmethods include, but are not limited to, antisense suppression (Smith etal., Nature 334:724-726 (1988)), co-suppression (Napoli et al., PlantCell 2:279-289 (1989)), ribozymes (Kohler et al., J. Mol. Biol.285:1935-1950 (1999)), combinations of sense and antisense (Waterhouseet al., Proc. Natl. Acad. Sci. USA 95:13959-13964 (1998)), promotersilencing (Park et al., Plant J. 9(2):183-194 (1996)), and DNA bindingproteins (Beerli et al., Proc. Natl. Acad. Sci. USA 95:14628-14633(1997); Liu et al., Proc. Natl. Acad. Sci. USA 94:5525-5530 (1998)).

[0007] Certain of these mechanisms are associated with nucleic acidhomology at the DNA or RNA level (Matzke et al., Current Opinion inGenetics and Development 11:221-227 (2001)). In plants, double-strandedRNA molecules can induce sequence-specific silencing. This phenomenon isoften referred to as double stranded RNA (“dsRNA”) in plants. Thisphenomenon has also been reported in Caenorhabditis elegans, where thisgene-specific silencing is often referred to as RNA interference or RNAi(Fire et al., Nature 391:806-811 (1988). Others have reported thisphenomenon in plants, fungi and animals (Sharp, Genes and Development13:139-141 (1999); Matzke et al., Current Opinion in Genetics andDevelopment 11:221-227 (2001); Cogoni and Macino, Current Opinion inGenetics and Development 10:638-643 (2000); Sharp, Genes and Development15:485-490 (2001); Waterhouse et al., Proc. Natl. Acad. Sci. USA95:13959-13964 (1988); Wesley et al., Plant J. 27:581-590 (2001);Grierson, WO 98/53083). Wesley et al. reported the design and use of twovectors, pHANNIBAL and pHELLSGATE, that can be used as gene silencingvectors (Wesley et al., supra). These vectors are reported to contain anintron sequence between the sense and antisense sequences where thesense and antisense sequences correspond to a target coding sequence,5′UTR or 3′UTR. By utilizing a non-target intron between the targetsense and antisense sequences, a higher proportion of silencedtransformants were obtained (Wesley et al., supra). Another strategy ofgene silencing with dsRNA involves a hairpin construct with an intronspacer (Smith et al., Nature 407:319-320 (2000)).

[0008] Other suppression strategies include, without limitation,antisense and sense suppression. See e.g. Fillatti in PCT WO 01/14538.

[0009] A desired plant phenotype may require the expression of one geneand the concurrent reduction of expression of another gene. Thus, thereexists a need to simultaneously over-express a polypeptide and suppress,or down-regulate, the expression of a second polypeptide in plants usinga single transgenic construct. Moreover, there exists a need tosimultaneously suppress or down-regulate the expression of more than onepolypeptide using a single construct.

SUMMARY OF THE INVENTION

[0010] The present invention includes and provides a nucleic acidmolecule comprising a first nucleic acid segment comprising apolypeptide encoding sequence and a second nucleic acid segmentcomprising a gene suppression sequence, wherein transcription of thenucleic acid molecule in a host cell results in expression of apolypeptide encoded by the polypeptide encoding sequence and suppressionof a gene in the host cell.

[0011] The present invention includes and provides a plant having anucleic acid molecule comprising a first nucleic acid segment comprisinga polypeptide encoding sequence and a second nucleic acid segmentcomprising a gene suppression sequence, wherein transcription of thenucleic acid molecule in a host cell results in expression of apolypeptide encoded by the polypeptide encoding sequence and suppressionof a gene in the host cell, wherein the first nucleic acid segment andthe second nucleic acid segment are operably linked to a single promotersequence.

[0012] The present invention also includes and provides a method ofsimultaneously altering the expression of more than one RNA moleculecomprising introducing into a plant cell a nucleic acid moleculecomprising a first nucleic acid segment comprising a polypeptideencoding sequence and a second nucleic acid segment comprising a genesuppression sequence, wherein transcription of the nucleic acid moleculein a host cell results in expression of a polypeptide encoded by thepolypeptide encoding sequence and suppression of a gene in the hostcell, wherein the first nucleic acid segment and the second nucleic acidsegment are operably linked to a single promoter sequence, and the firstnucleic acid segment and the second nucleic acid segment are expressed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a schematic of DNA construct elements in vectorpMON75565.

[0014]FIG. 2 is a schematic of DNA construct elements in vectorpMON75571.

[0015]FIGS. 3A and 3B are graphs depicting the percentage ofalpha-tocopherol in the total tocopherol content of Arabidopsis seedsfrom the R₂ generation of plants transformed with pMON75565 orpMON75571, respectively.

[0016]FIG. 4 is a graph representing the average seed oil and oleicfatty acid (18:1) levels in selected Arabidopsis seeds from the R₃generation of plants transformed with pMON75565.

[0017]FIGS. 5A and 5B are graphs depicting the total tocopherol levels(FIG. 5A) and percentage of alpha-tocopherol in the total tocopherolcontent (FIG. 5B) of Arabidopsis seeds from the R₃ generation of plantstransformed with pMON75565.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Description of Nucleic Acid Sequences

[0019] SEQ ID NO: 1 sets forth a nucleic acid sequence of a DNA moleculethat encodes a Gossypium hirsutum gamma-tocopherol methyltransferase.

[0020] SEQ ID NOs: 2 and 3 set forth nucleic acid sequences of primersfor use in amplifying a Gossypium hirsutum gamma methyl transferase.

[0021] SEQ ID NO: 4 is the 1405 nucleotide long DNA sequence of the RNAioperative element found at bases 3345-4947 of pMON75565. SEQ ID NO:4comprises in 5′ to 3′ direction a sense-oriented 3′UTR sequence fromArabidopsis thaliana FAD2 (bases 1-135) linked to a sense-orientedintron sequence with splice sites removed from Arabidopsis thaliana FAD2(bases 144-1275) linked to an antisense oriented 3′UTR sequence fromArabidopsis thaliana FAD2 (bases 1281-1405). FAD2 intron elementsessentially as in SEQ ID NO:4 are found within pMON75565 at bases3687-4818 and within SEQ ID:5 at bases 3396-4515.

[0022] SEQ ID NO:5 is the 8179 nucleotide long DNA sequence of thetransformation insertion element between Agrobacterium tumefaciensborder elements from vector pMON75565, i.e. the elements of a firsttranscription unit for simultaneously increasing expression of GMT anddecreasing expression of Δ12 desaturase by RNAi and a secondtranscription unit for a BAR marker.

[0023] SEQ ID NO:6 is the 7713 nucleotide long DNA sequence of thetransformation insertion element between Agrobacterium tumefaciensborder elements from vector pMON75571, i.e. the elements of a firsttranscription units for simultaneously increasing expression of GMT anddecreasing expression of Δ12 desaturase by inton sense suppression and asecond transcription unit for a BAR marker.

[0024] Definitions:

[0025] As used herein, “gene” refers to a nucleic acid sequence thatencompasses a 5′ promoter region associated with the expression of thegene product, any intron and exon regions and 3′ untranslated regions(“UTR”) associated with the expression of the gene product.

[0026] As used herein, “a transgenic plant” is any plant that stablyincorporates a transgene in a manner that facilitates transmission ofthat transgene from a plant by any sexual or asexual method.

[0027] As used herein, “transgene” refers to a nucleic acid sequenceassociated with the expression of a gene introduced to a cell of anorganism. A transgene includes, but is not limited to, a gene endogenousto or a gene not naturally occurring in the organism.

[0028] As used herein, “gene silencing” or “suppression” refers to thedown-regulation of gene expression by any method including, withoutlimitation, antisense suppression, sense suppression and sense intronsuppression. Such down-regulation can be a partial down-regulation.

[0029] As used herein, “a gene suppression sequence” is any nucleic acidsequence capable, when transcribed, of down-regulating gene expression.Such methods include but are not limited to antisense suppression, sensesuppression and sense intron suppression.

[0030] As used herein, “antisense suppression” refers to gene silencingthat is induced by the introduction of an antisense RNA molecule.

[0031] As used herein, “sense suppression” refers to gene silencing thatis induced by the introduction of a fragment of a gene in the senseorientation including, without limitation, a coding region or fragmentthereof.

[0032] As used herein, “sense intron suppression” refers to genesilencing that is induced by the introduction of a intron in the senseorientation or fragment thereof of a gene. Sense intron suppression isdescribed by Fillatti in PCT WO 01/14538 A2, the entirety of which isincorporated herein by reference.

[0033] When referring to proteins and nucleic acids herein, the use ofplain capitals, e.g., “GMT” or “FAD2,” indicates a reference to anenzyme, protein, polypeptide, or peptide, and the use of italicizedcapitals, e.g., “GMT” or “FAD2,” refers to nucleic acids, includingwithout limitation, genes, cDNAs, and mRNAs.

[0034] When referring to agents such as proteins and nucleic acidsherein, “derived” refers to obtaining a protein or nucleic acid from aknown protein or nucleic acid either directly (for example, by lookingat the sequence of a known protein or nucleic acid and preparing aprotein or nucleic acid having a sequence similar, at least in part, tothe sequence of the known protein or nucleic acid) or indirectly (forexample, by obtaining a protein or nucleic acid from an organism whichis related to a known protein or nucleic acid). Other methods of“deriving” a protein or nucleic acid from a known protein or nucleicacid are known to one of skill in the art.

[0035] When referring to nucleic acid constructs herein, it isunderstood that the construct may be in linear or circular form.

[0036] As used herein, “a nucleic acid segment” is a portion of a largernucleic acid molecule. Such nucleic acid segments can, for example,without limitation, comprise a polypeptide encoding sequence or a genesuppression sequence or both.

[0037] As used herein, “RNAi,” and “dsRNA,” refer to gene silencing thatis induced by the introduction of a double-stranded RNA molecule.

[0038] As used herein, a “dsRNA molecule” and an “RNAi molecule” bothrefer to a double-stranded RNA molecule capable, when introduced into acell or organism, of at least partially reducing the level of an mRNAspecies present in a cell or a cell of an organism.

[0039] As used herein, an “intron dsRNA molecule” and an “intron RNAimolecule” both refer to a double-stranded RNA molecule capable, whenintroduced into a cell or organism, of at least partially reducing thelevel of an mRNA species present in one or more cells where thedouble-stranded RNA molecule exhibits sufficient identity to an intronof a gene present in the cell or organism to reduce the level of an mRNAcontaining that intron sequence.

[0040] The term “non-coding” refers to sequences of nucleic acidmolecules that do not encode part or all of an expressed protein.Non-coding sequences include but are not limited to introns, promoterregions, 3′ untranslated regions (“3′UTR”), and 5′ untranslated regions(“5′UTR”).

[0041] The term “intron” as used herein refers to the normal sense ofthe term as meaning a segment of a nucleic acid molecule, usually DNA,that does not encode part of or all of an expressed protein, and which,in endogenous conditions, is transcribed into RNA molecules, but whichis spliced out of the endogenous RNA before the RNA is translated into aprotein.

[0042] The term “exon” as used herein refers to the normal sense of theterm as meaning a segment of nucleic acid molecules, usually DNA, whichencodes part of or all of an expressed protein.

[0043] As used herein, a promoter that is “operably linked” to one ormore nucleic acid sequences is capable of driving expression of one ormore nucleic acid sequences, including multiple coding or non-codingnucleic acid sequences arranged in a polycistronic configuration orconstruct.

[0044] As used herein, a “plant promoter” includes, without limitation,a plant viral promoter and a synthetic, chimeric or hybrid promoter,which is a single transcriptional unit, capable of functioning in aplant cell to promote the expression of an mRNA.

[0045] A “polycistronic configuration” or “polycistronic construct” is aconfiguration which comprises nucleic acid sequences of more than onegene. It is understood that within a “polycistronic configuration” theremay be sequences that correspond to exons, introns or both, and a“polycistronic configuration” might, for example without limitation,contain sequences that correspond to one or more exons from one gene andone or more introns from a second gene.

[0046] As used herein, a “polycistronic gene” or “polycistronic mRNA” isany gene or mRNA that contains nucleic acid sequences which correspondto nucleic acid sequences of more than one gene. It is understood thatsuch polycistronic genes or mRNAs may contain sequences that correspondto exons, introns or both and that a recombinant polycistronic gene ormRNA might, for example without limitation, contain sequences thatcorrespond to one or more exons from one gene and one or more intronsfrom a second gene.

[0047] As used herein, “physically linked” nucleic acid sequences arenucleic acid sequences that are found on a single nucleic acid molecule.

[0048] As used herein, “expression” refers to the process oftranscription and translation.

[0049] As used herein, “simultaneous expression” of more than one agentsuch as an mRNA or protein refers to the expression of an agent at thesame time as another agent. Such expression may only overlap in part andmay also occur in different tissues or at different levels.

[0050] As used herein, “simultaneously altering expression” of more thanone agent such as an mRNA or protein refers to altering the expressionof an agent at the same time as altering the expression of anotheragent. Such expression of the more than one agent may be altered indifferent tissues or at different levels.

[0051] As used herein, “coexpression” of more than one agent such as anmRNA or protein refers to the simultaneous expression of an agent at thesame time and in the same cell or tissue as another agent.

[0052] As used herein, “coordinated expression” of more than one agent”refers to the coexpression of more than one agent when the expression ofsuch agents is carried out utilizing a shared or identical promoter.

[0053] As used herein, an “at least partially enhanced” or an“increased” level of an agent such as a protein or mRNA is at leastpartially enhanced or increased if the level of that agent is increasedrelative to the level that that agent is present in a cell, tissue,plant or organism with a similar genetic background but lacking anintroduced nucleic acid molecule encoding the protein or mRNA.

[0054] As used herein, a “polypeptide” comprises fifteen or greateramino acid residues.

[0055] As used herein, a “peptide” contains 14 or fewer amino acidresidues.

[0056] As used herein, an “enhanced” level of an agent such as aprotein, polypeptide, peptide, or mRNA is enhanced if the level of thatagent is increased at least 25% relative to the level that that agent ispresent in a cell, tissue, plant or organism with a similar geneticbackground but lacking an introduced nucleic acid molecule encoding theprotein or mRNA.

[0057] As used herein, the level of an agent such as a protein,polypeptide, peptide, or mRNA is “substantially enhanced” if the levelof that agent is increased at least 75% relative to the level that thatagent is present in a cell, tissue, plant or organism with a similargenetic background but lacking an introduced nucleic acid moleculeencoding the protein or mRNA.

[0058] As used herein, “a reduction” of the level of an agent such as aprotein, polypeptide, peptide, or mRNA means that the level is decreasedrelative to a cell, tissue, plant or organism with a similar geneticbackground but lacking a nucleic acid sequence capable of reducing theagent.

[0059] As used herein, “at least a partial reduction” of the level of anagent such as a protein, polypeptide, peptide, or mRNA means that thelevel is decreased at least 25% relative to a cell, tissue, plant ororganism with a similar genetic background but lacking a nucleic acidsequence capable of reducing the agent.

[0060] As used herein, “a substantial reduction” of the level of anagent such as a protein, polypeptide, peptide, or mRNA means that thelevel is decreased relative to a cell, tissue, plant or organism with asimilar genetic background but lacking a nucleic acid sequence capableof reducing the agent, where the decrease in the level of the agent isat least 75%.

[0061] As used herein, “an effective elimination” of an agent such as aprotein, polypeptide, peptide, or mRNA is relative to a cell, tissue,plant or organism with a similar genetic background but lacking anucleic acid sequence capable of decreasing the agent, where thedecrease in the level of the agent is greater than 95%.

[0062] As used herein, “heterologous” means not naturally occurringtogether.

[0063] As used herein, “an endogenous gene” is any gene that is notintroduced into a host by transformation or transfection.

[0064] As used herein, “total oil level” refers to the total aggregateamount of fatty acid without regard to the type of fatty acid.

[0065] As used herein, an “altered seed oil composition” refers to aseed composition in which the relative percentages of the types of fattyacids are altered.

[0066] As used herein, any range set forth is inclusive of the endpoints of the range unless otherwise stated.

[0067] Agents of the invention will preferably be “biologically active”with respect to either a structural attribute, such as the capacity of anucleic acid molecule to hybridize to another nucleic acid molecule, orthe ability of a protein to be bound by an antibody (or to compete withanother molecule for such binding). Alternatively, such biologicalactivity may be catalytic and thus involve the capacity of the agent tomediate a chemical reaction or response.

[0068] Agents will preferably be “substantially purified.” The term“substantially purified,” as used herein, refers to a molecule separatedfrom substantially all other molecules normally associated with it inits native environmental conditions. More preferably a substantiallypurified molecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than 60% free, greaterthan 75% free, preferably greater than 90% free, and most preferablygreater than 95% free from the other molecules (exclusive of solvent)present in the natural mixture. The term “substantially purified” is notintended to encompass molecules present in their native environmentalconditions.

[0069] Agents of the invention may also be recombinant. As used herein,the term “recombinant” means any agent (e.g., including but not limitedto DNA, RNA, peptide), that is, or results, however indirectly, fromhuman manipulation of a nucleic acid molecule or peptide.

[0070] Agents of the invention may be labeled with reagents thatfacilitate detection of the agent (e.g., fluorescent labels, Prober etal., Science 238:336-340 (1987); Albarella et al., EP 144914; chemicallabels, Sheldon et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S.Pat. No. 4,563,417; modified bases, Miyoshi et al., EP 119448).

[0071] As used herein, “% identity” is determined using the followingparameters and algorithm: Smith Waterman algorithm is used to determineidentity. Parameters for polypeptide sequence comparison typicallyinclude the following: Algorithm: Needleman and Wunsch, J. Mol. Biol.48:443-453 (1970). Comparison matrix: BLOSSUM62 from Hentikoff andHentikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992). GapPenalty: 12; Gap Length Penalty: 4. A program that can be used withthese parameters is publicly available as the “gap” program fromGenetics Computer Group (“GCG”), Madison, Wis. The above parametersalong with no penalty for end gap are the default parameters for peptidecomparisons. Parameters for nucleic acid molecule sequence comparisonare the following: Algorithm: Needleman and Wunsch, J. Mol. Bio.48:443-453 (1970). Comparison matrix: matches—+10, mismatches=0; GapPenalty: 50; Gap Length Penalty: 3. “% identity” is determined using theabove parameters as the default parameters for nucleic acid moleculesequence comparisons and the “gap” program from GCG, version 10.2.

[0072] As used herein, a gamma-tocopherol methyltransferase (alsoreferred to as GMT, γ-GMT, γ-MT, γ-TMT or gamma-methyltransferase) isany polypeptide that is capable of specifically catalyzing theconversion of γ-tocopherol into α-tocopherol. In certain plant speciessuch as soybean, GMT can also catalyze the conversion of δ-tocopherol toβ-tocopherol. In other plants, GMT can also catalyze the conversion ofδ-tocotrienol to β-tocotrienol.

[0073] As used herein, a “FAD2”, “Δ12 desaturase” or “omega-6desaturase” gene is a gene that encodes an enzyme capable of catalyzingthe insertion of a double bond into a fatty acyl moiety at the twelfthposition counted from the carboxyl terminus.

[0074] Nucleic Acid Molecules, Constructs and Vectors

[0075] Vector systems suitable for introducing transforming DNA into ahost plant cell include, but are not limited to, binary bacterialartificial chromosome (BIBAC) vectors (Hamilton et al., Gene 200:107-116(1997)); RNA viral vectors (Della-Cioppa et al., Ann. N.Y. Acad. Sci.792 (Engineering Plants for Commercial Products and Applications):57-61(1996)); plant selectable YAC (Yeast Artificial Chromosome) vectors suchas those described in Mullen et al., Molecular Breeding 4:449-457(1988); cosmids; and bacterial artificial chromosomes (BACs), and suchvector systems can be utilized with nucleic acid molecules of thepresent invention. In one aspect of the invention such vectors contain anucleic acid molecule comprising a first nucleic acid segment comprisinga polypeptide encoding sequence and a second nucleic acid segmentcomprising a gene suppression sequence, wherein transcription of saidnucleic acid molecule in a host cell results in expression of apolypeptide encoded by the polypeptide encoding sequence and suppressionof a gene in the host cell. In one aspect, the first nucleic acid andthe second nucleic acid segment are operably linked to a single promotersequence. A second nucleic acid segment may be expressed, for example,without limitation, as a dsRNA molecule, an RNAi molecule, an introndsRNA molecule, or an intron RNAi molecule. In an aspect of the presentinvention, such first nucleic acid segment and second nucleic acidsegment can be expressed, coexpressed, or coordinately expressed in ahost cell and, upon expression, the RNA encoded by the second nucleicacid segment is suppressed.

[0076] A. Promoter

[0077] In an aspect of the present invention, nucleic acid molecules,constructs or vectors contain a promoter that is operably linked to oneor more nucleic acid sequences. Any promoter that functions in a plantcell to cause the production of an mRNA molecule, such as thosepromoters described herein, without limitation, can be used. In apreferred embodiment, the promoter is a plant promoter.

[0078] A number of promoters that are active in plant cells have beendescribed in the literature. These include, but are not limited to, thenopaline synthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci.USA 84:5745-5749 (1987)), the octopine synthase (OCS) promoter (which iscarried on tumor-inducing plasmids of Agrobacterium tumefaciens), thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Spromoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987)), and theCaMV 35S promoter (Odell et al., Nature 313:810-812 (1985)), the figwortmosaic virus 35S-promoter (U.S. Pat. No. 5,378,619), the light-induciblepromoter from the small subunit of ribulose-1,5-bis-phosphatecarboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl.Acad. Sci. USA 84:6624-6628 (1987)), the sucrose synthase promoter (Yanget al., Proc. Natl. Acad. Sci. USA 87:4144-4148 (1990)), the R genecomplex promoter (Chandler et al., Plant Cell 1:1175-1183 (1989)) andthe chlorophyll a/b binding protein gene promoter. These promoters havebeen used to create DNA constructs that have been expressed in plants(See, e.g., PCT WO 84/02913). The CaMV 35S promoters are preferred foruse in plants. See also U.S. Pat. No. 6,437,217, which discloses a maizeRS81 promoter; U.S. Pat. No. 5,641,876, which discloses a rice actinpromoter; U.S. Pat. No. 6,426,446, which discloses a maize RS324promoter; U.S. Pat. No. 6,429,362, which discloses a maize PR-1promoter; U.S. Pat. No. 6,232,526, which discloses a maize A3 promoter;and U.S. Pat. No. 6,177,611, which discloses constitutive maizepromoter, all of which are incorporated by reference. The rice actin 1promoter with a rice actin intron is especially useful in the practiceof the present invention.

[0079] Particularly preferred promoters can also be used to express anucleic acid molecule of the present invention in seeds or fruits.Indeed, in a preferred embodiment, the promoter used is a seed specificpromoter. Examples of such promoters include the 5′ regulatory regionsfrom such genes as napin (Kridl et al., Seed Sci. Res. 1:209:219(1991)), phaseolin (Bustos et al., Plant Cell 1(9):839-853 (1989)),soybean trypsin inhibitor (Riggs et al., Plant Cell 1(6):609-621(1989)), ACP (Baerson et al., Plant Mol. Biol. 22(2):255-267 (1993)),stearoyl-ACP desaturase (Slocombe et al., Plant Physiol. 104(4):167-176(1994)), soybean a′ subunit of b-conglycinin (soy 7 s promoter, (Chen etal., Proc. Natl. Acad. Sci. USA 83:8560-8564 (1986))), fatty acidelongation (FAE1) promoter (PCT WO 01/11061), and oleosin (see, forexample, Hong et al., Plant Mol. Biol. 34(3):549-555 (1997)). Furtherexamples include the promoter for β-conglycinin (Chen et al., Dev.Genet. 10: 112-122 (1989)). Preferred promoters for expression in theseed are 7 S and napin promoters.

[0080] Also included are the zein promoters, which are a group ofstorage proteins found in corn endosperm. Genomic clones for zein geneshave been isolated (Pedersen et al., Cell 29:1015-1026 (1982); Russellet al., Transgenic Res. 6(2):157-168 (1997)) and the promoters fromthese clones, including the 15 kD, 16 kD, 19 kD, 22 kD, and 27 kD genes,could also be used. Other promoters known to function, for example incorn, include the promoters for the following genes: waxy, Brittle,Shrunken 2, Branching enzymes I and II, starch synthases, debranchingenzymes, oleosins, glutelins and sucrose synthases. A particularlypreferred promoter for corn endosperm expression is the promoter for theglutelin gene from rice, more particularly the Osgt-1 promoter (Zheng etal., Mol. Cell Biol. 13:5829-5842 (1993)). Examples of promoterssuitable for expression in wheat include those promoters for the ADPglucose pyrosynthase (ADPGPP) subunits, the granule bound and otherstarch synthase, the branching and debranching enzymes, theembryogenesis-abundant proteins, the gliadins and the glutenins.Examples of such promoters in rice include those promoters for theADPGPP subunits, the granule bound and other starch synthase, thebranching enzymes, the debranching enzymes, sucrose synthases and theglutelins. A particularly preferred promoter is the promoter for riceglutelin, Osgt-1. Examples of such promoters for barley include thosefor the ADPGPP subunits, the granule bound and other starch synthase,the branching enzymes, the debranching enzymes, sucrose synthases, thehordeins, the embryo globulins and the aleurone specific proteins.

[0081] Tissue-specific expression of a protein of the present inventionis a particularly preferred embodiment. The tissue-specific promotersthat can be used include the chloroplast glutamine synthetase GS2promoter from pea (Edwards et al., Proc. Natl. Acad. Sci. USA87:3459-3463 (1990)), the chloroplast fructose-1,6-biphosphatase(FBPase) promoter from wheat (Lloyd et al., Mol. Gen. Genet. 225:209-216(1991)), the nuclear photosynthetic ST-LS1 promoter from potato(Stockhaus et al., EMBO J 8:2445-2451 (1989)), the serine/threoninekinase (PAL) promoter and the glucoamylase (CHS) promoter fromArabidopsis thaliana. Also reported to be active in photosyntheticallyactive tissues are the ribulose-1, 5-bisphosphate carboxylase (rbcS)promoter from eastern larch (Larix laricina), the promoter for the cabgene, cab6, from pine (Yamamoto et al., Plant Cell Physiol. 35:773-778(1994)), the promoter for the Cab-1 gene from wheat (Fejes et al., PlantMol. Biol. 15:921-932 (1990)), the promoter for the CAB-1 gene fromspinach (Lubberstedt et al., Plant Physiol. 104:997-1006 (1994)), thepromoter for the cab1R gene from rice (Luan et al., Plant Cell 4:971-981(1992)), the pyruvate, orthophosphate dikinase (PPDK) promoter from corn(Matsuoka et al., Proc. Natl. Acad. Sci. USA 90: 9586-9590 (1993)), thepromoter for the tobacco Lhcb1*2 gene (Cerdan et al., Plant Mol. Biol.33:245-255 (1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporterpromoter (Truernit et al., Planta. 196:564-570 (1995)) and the promoterfor the thylakoid membrane proteins from spinach (psaD, psaF, psaE, PC,FNR, atpC, atpD, cab, rbcS). Other promoters for the chlorophylla/b-binding proteins may also be utilized in the invention, such as thepromoters for LhcB gene and PsbP gene from white mustard (Sinapis alba;Kretsch et al., Plant Mol. Biol. 28:219-229 (1995)).

[0082] A number of promoters for genes with tuber-specific ortuber-enhanced expression are known and can be used, including the classI patatin promoter (Bevan et al., EMBO J 8:1899-1906 (1986); Jeffersonet al., Plant Mol. Biol. 14:995-1006 (1990)), the promoter for thepotato tuber ADPGPP genes, both the large and small subunits, thesucrose synthase promoter (Salanoubat and Belliard, Gene 60:47-56(1987), Salanoubat and Belliard, Gene 84:181-185 (1989)), the promoterfor the major tuber proteins including the 22 kd protein complexes andprotease inhibitors (Hannapel, Plant Physiol. 101:703-704 (1993)), thepromoter for the granule-bound starch synthase gene (GBSS) (Visser etal., Plant Mol. Biol. 17:691-699 (1991)) and other class I and IIpatatins promoters (Koster-Topfer et al., Mol. Gen. Genet. 219:390-396(1989); Mignery et al., Gene. 62:27-44 (1988)).

[0083] Root specific promoters may also be used. An example of such apromoter is the promoter for the acid chitinase gene (Samac et al.,Plant Mol. Biol. 25:587-596 (1994)). Expression in root tissue couldalso be accomplished by utilizing the root specific subdomains of theCaMV35S promoter that have been identified (Lam et al., Proc. Natl.Acad. Sci. USA 86:7890-7894 (1989)). Other root cell specific promotersinclude those reported by Conkling et al. (Conkling et al., PlantPhysiol. 93:1203-1211 (1990)).

[0084] The promoters used in the nucleic acid constructs of the presentinvention may be modified, if desired, to affect their controlcharacteristics. Promoters can be derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters may be altered to contain multiple “enhancer sequences” toassist in elevating gene expression. Such enhancers are known in theart. By including an enhancer sequence with such constructs, theexpression of the selected protein may be enhanced. These enhancersoften are found 5′ to the start of transcription in a promoter thatfunctions in eukaryotic cells, but can often be inserted in the forwardor reverse orientation 5′ or 3′ to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Deemed to beparticularly useful as enhancers are the 5′ introns of the rice actin 1and rice actin 2 genes. Examples of other enhancers which could be usedin accordance with the invention include elements from the CaMV 35Spromoter, octopine synthase genes, the maize alcohol dehydrogenase gene,the maize shrunken 1 gene and promoters from non-plant eukaryotes.

[0085] Where an enhancer is used in conjunction with a promoter for theexpression of a selected protein, it is believed that it will bepreferred to place the enhancer between the promoter and the start codonof the selected coding region. However, one also could use a differentarrangement of the enhancer relative to other sequences and stillrealize the beneficial properties conferred by the enhancer. Forexample, the enhancer could be placed 5′ of the promoter region, withinthe promoter region, within the coding sequence (including within anyother intron sequences which may be present), or 3′ of the codingregion.

[0086] In addition to introns with enhancing activity, other types ofelements can influence gene expression. For example, untranslated leadersequences predicted to enhance gene expression as well as “consensus”and preferred leader sequences have been identified. Preferred leadersequences are contemplated to include those which have sequencespredicted to direct optimum expression of the attached coding region,i.e., to include a preferred consensus leader sequence which mayincrease or maintain mRNA stability and prevent inappropriate initiationof translation. The choice of such sequences will be known to those ofskill in the art in light of the present disclosure. Sequences that arederived from genes that are highly expressed in plants, and in maize inparticular, will be most preferred. For example, sequences derived fromthe small subunit of ribulose bisphosphate carboxylase (RUBISCO).

[0087] In general it is preferred to introduce heterologous DNArandomly, i.e. at a non-specific location, in the genome. In specialcases it may be useful to target heterologous nucleic acid insertion inorder to achieve site specific integration, e.g. to replace an existinggene in the genome. In some other cases it may be useful to target aheterologous nucleic acid integration into the genome at a predeterminedsite from which it is known that gene expression occurs. Several sitespecific recombination systems exist which are known to function inplants including cre-lox as disclosed in U.S. Pat. No. 4,959,317 andFLP-FRT as disclosed in U.S. Pat. No. 5,527,695.

[0088] Additional promoters that may be utilized are described, forexample, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858;5,608,144; 5,614,399; 5,633,441; 5,633,435; and 4,633,436. In addition,a tissue specific enhancer may be used (Fromm et al., Plant Cell1:977-984 (1989)).

[0089] B. Nucleic Acid Molecules

[0090] In an aspect of the invention, the nucleic acid moleculecomprises a nucleic acid sequence, which when introduced into a cell ororganism, is capable of simultaneously overexpressing, expressing,coexpressing or coordinately expressing one or more RNA molecules toproduce one or more proteins, fragments thereof, polypeptides, orpeptides while expressing one or more other RNA molecules capable ofsuppressing the level of one or more RNA molecules expressed in the cellor organism.

[0091] In this aspect of the present invention any protein, fragmentthereof, polypeptide, or peptide can be expressed and any RNA moleculecan be suppressed. Nucleic acid sequences encoding such proteins,fragments thereof, polypeptides, and peptides as well as nucleic acidsequences useful in the suppression of one or more mRNA moleculesexpressed in the cell or organism can be derived, for example, withoutlimitation, from a gene, fragment thereof, cDNA, fragment thereof, etc.

[0092] A gene of the present invention can be any gene, whetherendogenous or introduced. Nucleic acid sequences of such genes can bederived from a multitude of sources, including, without limitation,databases such as EMBL and Genbank found at www-ebi.ac.uk/swisprot/;www-expasy.ch/; www-embl.heidelberg.de/; and www-ncbi.nlm.nih.gov.Nucleic acid sequences of such genes can also be derived, withoutlimitation, from sources such as the GENSCAN program found athttp-genes.mit.edu/GENSCAN.html. In a further embodiment, additionalgenes may be obtained by any method by which additional genes may beidentified. In a preferred embodiment, an additional gene may beobtained by screening a genomic library with a probe of known genesequences. The gene may then be cloned and confirmed. Additional genesmay, for example without limitation, be amplified by polymerase chainreaction (PCR) and used in an embodiment of the present invention. Inaddition, other nucleic acid sequences of genes will be apparent to oneof ordinary skill in the art.

[0093] Any of a variety of methods may be used to obtain one or moregenes. Automated nucleic acid synthesizers may be employed for thispurpose, and to make a nucleic acid molecule that has a sequence alsofound in a cell or organism. In lieu of such synthesis, nucleic acidmolecules may be used to define a pair of primers that can be used withthe PCR to amplify and obtain any desired nucleic acid molecule orfragment of a first gene.

[0094] In a preferred aspect, the gene, mRNA or protein is a non-viralgene, mRNA or protein. In another preferred aspect, the gene, RNA orprotein is an endogenous gene, RNA or protein. In a preferred aspect, agene is a GMT gene. A preferred GMT gene of the present invention is aplant or cyanobacterial GMT, more preferably a GMT that is also found inan organism selected from the group consisting of Arabidopsis, rice,corn, cotton, cuphea, oilseed rape, tomato, soybean, marigold, sorghum,and leek, most preferably a GMT that is also found in an organismselected from the group consisting of Arabidopsis thaliana, Oryzasativa, Zea mays, Gossypium hirsutum, Cuphea pulcherrima, Brassicanapus, Lycopersicon esculentum, Glycine max, Tagetes erecta, and Liliumasiatic. Representative sequences for GMT genes include, withoutlimitation, those set forth in U.S. patent application Ser. No.10/219,810, filed on Aug. 16, 2002.

[0095] In an aspect, another preferred gene of the present invention isa FAD2 gene. Representative sequences for FAD2 include, withoutlimitation, those set forth in U.S. application Ser. No. 10/176,149,filed Jun. 21, 2002, and U.S. patent application Ser. No. 09/638,508,filed Aug. 11, 2000, and U.S. Provisional Application Serial No.60/151,224, filed Aug. 26, 1999, and U.S. Provisional Application SerialNo. 60/172,128, filed Dec. 17, 1999. In a preferred aspect a GMT proteinis expressed and the expression of a FAD2 protein is suppressed.

[0096] In an aspect of the present invention, a nucleic acid moleculecomprising a first nucleic acid segment comprising a polypeptideencoding sequence and a second nucleic acid segment comprising a genesuppression sequence, wherein transcription of the nucleic acid moleculein a host cell results in expression of a polypeptide encoded by thepolypeptide encoding sequence and suppression of a gene in said hostcell, where the first nucleic acid segment and the second nucleic acidsegment are operably linked to a single promoter sequence.

[0097] In a preferred aspect of the present invention the nucleic acidmolecule further comprises nucleotide sequences encoding a plastidtransit peptide operably fused to a nucleic acid molecule of the presentinvention that encodes a protein, fragment thereof, polypeptide, orpeptide.

[0098] A nucleic acid molecule or protein, fragment thereof,polypeptide, or peptide of the present invention may differ in eithernucleic acid or amino acid sequence from a gene or its translatedproduct but nonetheless share a percentage identity with a nucleic acidor amino acid sequence from a gene. “Identity,” as is well understood inthe art, is a relationship between two or more polypeptide sequences ortwo or more nucleic acid molecule sequences, as determined by comparingthe sequences. In the art, “identity” also means the degree of sequencerelatedness between polypeptide or nucleic acid molecule sequences, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods.

[0099] In another aspect, the nucleic acid sequence of the nucleic acidmolecules of the present invention can comprise sequences that differfrom those encoding a protein, fragment thereof, polypeptide, or peptidedue to the fact that a protein, fragment thereof, polypeptide, orpeptide can have one or more conservative amino acid changes, andnucleic acid sequences coding for the polypeptide can therefore havesequence differences.

[0100] It is well known in the art that one or more amino acids in anative sequence can be substituted with other amino acid(s), the chargeand polarity of which are similar to that of the native amino acid,i.e., a conservative amino acid substitution. Hydropathic index of aminoacids may also be considered when making amino acid changes. Theimportance of the hydropathic amino acid index in conferring interactivebiological function on a protein is generally understood in the art(Kyte and Doolittle, J. Mol. Biol. 157:105-132 (1982)). It is alsounderstood in the art that the substitution of like amino acids can bemade effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the protein. In making such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within +1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0101] Due to the degeneracy of the genetic code, different nucleotidecodons may be used to code for a particular amino acid. A host celloften displays a preferred pattern of codon usage. Structural nucleicacid sequences are preferably constructed to utilize the codon usagepattern of the particular host cell. This generally enhances theexpression of the structural nucleic acid sequence in a transformed hostcell. Any of the above-described nucleic acid and amino acid sequencesmay be modified to reflect the preferred codon usage of a host cell ororganism in which they are contained. Modification of a structuralnucleic acid sequence for optimal codon usage in plants is described inU.S. Pat. No. 5,689,052.

[0102] Preferred embodiments of the invention include nucleic acidmolecules that comprise a first, second or both nucleic acid segment(s),which is at least 50%, 60%, or 70% identical over its entire length to aplant gene. More preferable are first, second or both nucleic acidsegments which comprise a region that is at least 80% or at least 85%identical over its entire length to a plant gene. In this regard firstand second nucleic acid segments at least 90% identical over theirentire length are particularly preferred, those at least 95% identicalare especially preferred. Further, those with at least 97% identity arehighly preferred and those with at least 98% and at least 99% identityare particularly highly preferred, with those exhibiting 100% identitybeing the most highly preferred.

[0103] A subset of the first or second nucleic acid segment of thenucleic acid molecules of the invention includes fragment nucleic acidmolecules. Fragment nucleic acid molecules may consist of significantportion(s) of, or indeed most of, a plant gene. Alternatively, fragmentsmay comprise smaller oligonucleotides, having from about 15 to about 400contiguous nucleotide residues and more preferably, about 15 to about 45contiguous nucleotide residues, about 20 to about 45 contiguousnucleotide residues, about 15 to about 30 contiguous nucleotideresidues, about 21 to about 30 contiguous nucleotide residues, about 21to about 25 contiguous nucleotide residues, about 21 to about 24contiguous nucleotide residues, about 19 to about 25 contiguousnucleotide residues, or about 21 contiguous nucleotides. In a preferredembodiment, a fragment shows 100% identity to a region of a plant gene.In another preferred embodiment, a fragment comprises a portion of alarger nucleic acid sequence. In another aspect, a fragment nucleic acidmolecule has a nucleic acid sequence that has at least 15, 25, 50, or100 contiguous nucleotides of a nucleic acid molecule of the presentinvention. In a preferred embodiment, a nucleic acid molecule has anucleic acid sequence that has at least 15, 25, 50, or 100 contiguousnucleotides of a plant gene.

[0104] It is understood that a nucleic acid of the present invention canbe in either orientation and that such molecules can be in a sense orantisense orientation.

[0105] A first nucleic acid segment can be physically linked to or partof a polycistronic construct with a second nucleic acid segment. Nucleicacid sequences within a first or second nucleic acid segment can bephysically linked to or part of a polycistronic construct with othernucleic acid segments. A promoter can be physically linked to or part ofa polycistronic construct with a first nucleic segment and secondnucleic acid segment. Such polycistronic constructs can be capable ofexpressing a polycistronic mRNA.

[0106] i. First Nucleic Acid Segment Capable of Being Transcribed as Oneor More RNAs

[0107] A first nucleic acid segment can be any nucleic acid sequencethat is capable of being transcribed and expressed as an mRNA. In anaspect, the nucleic acid sequence corresponds to a nucleic acid sequencethat is also found in a naturally occurring gene or part of a gene suchas a transcribed segment of a gene. Such a gene can be any gene from anyorganism. In a preferred aspect the gene is from a plant. In anotherpreferred aspect the gene is from a microorganism. An illustrative geneis a GMT gene. A first nucleic acid segment which is transcribed andexpressed as an mRNA can be translated into a protein, fragment thereof,polypeptide, or peptide. In one aspect the proteins, fragments thereof,polypeptides, or peptides are also endogenous to the host. In anotheraspect the proteins, fragments thereof, polypeptides, or peptides arenot normally found in the plant. In a further aspect the amino acidsequence of the proteins, fragments thereof, polypeptides, or peptidesare not found in a non-transformed host.

[0108] It is also understood that a first nucleic acid segment cancontain sequences that encode for more than one protein, fragmentthereof, polypeptide, or peptide. In this aspect, the proteins,fragments thereof, polypeptides, or peptides may be a combination ofproteins, fragments thereof, polypeptides, or peptides endogenous to thehost, not normally found in the plant, or not found in a non-transformedhost. In this aspect, a first nucleic acid segment can encode for two,three, four, five, or more than five proteins, fragments thereof,polypeptides, or peptides.

[0109] ii. Second Nucleic Acid Sequence Capable of Suppressing One orMore RNAs

[0110] A second nucleic acid segment can be any nucleic acid sequencewhich, when introduced into a cell or organism, is capable ofeffectively eliminating, substantially reducing, at least partiallyreducing or reducing the level of an mRNA transcript or protein encodedby a gene. In an aspect of the present invention, a gene is anendogenous gene. In an aspect of the present invention, a gene is aplant gene. An illustrative gene is a FAD2 gene.

[0111] It is also understood that a second nucleic acid segment can beany nucleic acid sequence, which, when introduced into a cell ororganism, is capable of effectively eliminating, substantially reducing,at least partially reducing or reducing the level of one, two, three,four, five, or more mRNAs. It also understood in this aspect that anindividual mRNA may be suppressed by different methodologies, forexample RNAi and antisense suppression.

[0112] In an aspect of the invention, the second nucleic acid sequenceof the present invention, which is preferably a dsRNA construct,preferably a sense RNA construct, or preferably an antisense RNAconstruct, is capable of providing at least a partial reduction, morepreferably a substantial reduction, or most preferably effectiveelimination of another agent such as a protein or mRNA. In an aspect ofthe present invention, the other agent is a FAD2 protein or mRNA encodedby a FAD2 gene.

[0113] In another aspect, the level of one or more agents is reduced, atleast partially reduced, substantially reduced or effectively eliminatedwhile the level of one or more simultaneously, co-expressed orcoordinately expressed agents is at least partially enhanced, at leastenhanced, or substantially enhanced.

[0114] In a further embodiment, a nucleic acid molecule, when introducedinto a cell or organism, selectively increases the level of a firstprotein or RNA transcript or both encoded by a first gene and at thesame time reduces the level of a second protein, transcript or bothencoded by a second gene, and also alters the alpha-tocopherol content,the oil composition, and the oil level of the cell or organism.

[0115] Multiple methodologies can be used to effectively eliminate,substantially reduce, or at least partially reduce the level of an mRNAtranscript or protein encoded by a gene. Such methods can result in genespecific silencing or in the silencing of multiple genes. Examples ofsuch gene silencing include, without limitation, those induced by theintroduction of a double-stranded RNA molecule, antisense, and senseRNA.

[0116] In another aspect, a second nucleic acid segment can be anynucleic acid sequence which, when introduced into a cell or organism, iscapable of effectively eliminating, substantially reducing, at leastpartially reducing or reducing the level of two, three, four, five, ormore than five mRNA transcripts or proteins encoded by a gene.

[0117] a. dsRNA

[0118] Double-stranded molecules which can be used for gene silencinginclude dsRNA molecules that comprise nucleic acid sequencescorresponding to a nucleic acid sequence found in a transcript. Suchnucleic acid sequences include, without limitation, nucleic acidsequences that encode for a protein, fragment thereof, polypeptide, orpeptide, and those that correspond to transcribed introns, transcribed3′ untranslated regions (UTRs), and transcribed 5′ UTRs.

[0119] One subset of the second nucleic acid sequence of the nucleicacid molecules of the invention is a nucleic acid sequence which isexpressed as a double-stranded RNA which comprises (1) a first RNAfragment that exhibits identity to a transcribed region of a second genewhich is to be suppressed, and (2) a second RNA capable of forming adouble-stranded RNA molecule with the first RNA. The first RNA fragmentmay consist of significant portion(s) of, or indeed most of, a plantgene which is to be suppressed.

[0120] In an aspect, a nucleic acid molecule of the present inventioncomprises a nucleic acid sequence which exhibits sufficient homology toone or more plant introns from a second plant gene, which whenintroduced into a plant cell or plant as a dsRNA construct, is capableof effectively eliminating, substantially reducing, or at leastpartially reducing the level of an mRNA transcript or protein encoded bythe gene from which the intron(s) was derived.

[0121] In an aspect, a nucleic acid molecule of the present inventioncomprises a nucleic acid sequence which exhibits sufficient homology toone or more plant exons from a second plant gene, which when introducedinto a plant cell or plant as a dsRNA construct, is capable ofeffectively eliminating, substantially reducing, or at least partiallyreducing the level of an mRNA transcript or protein encoded by the genefrom which the exon(s) was derived.

[0122] In an aspect, a nucleic acid molecule of the present inventioncomprises a nucleic acid sequence which exhibits sufficient homology toone or more plant transcribed 3′ UTR(s) from a second plant gene, whichwhen introduced into a plant cell or plant as a dsRNA construct, iscapable of effectively eliminating, substantially reducing, or at leastpartially reducing the level of an mRNA transcript or protein encoded bythe gene from which the 3′ UTR(s) was derived.

[0123] In an aspect, a nucleic acid molecule of the present inventioncomprises a nucleic acid sequence which exhibits sufficient homology toone or more plant transcribed 5′ UTR(s) from a second plant gene, whichwhen introduced into a plant cell or plant as a dsRNA construct, iscapable of effectively eliminating, substantially reducing, or at leastpartially reducing the level of an mRNA transcript or protein encoded bythe gene from which the 5′ UTR(s) was derived.

[0124] In another preferred aspect, a dsRNA construct contains exonsequences, but the exon sequences do not correspond to a sufficient partof a plant exon to be capable of effectively eliminating, substantiallyreducing, or at least partially reducing the level of an mRNA transcriptor protein encoded by a second gene from which the exon was derived.Strategies of suppressing gene expression with dsRNA constructs includethat set forth in U.S. Provisional Patent Application Serial No.60/390,186, filed on Jun. 9, 2000.

[0125] b. Antisense Suppression

[0126] Antisense molecules which can be used for gene silencing includeany molecules that comprise nucleic acid sequences corresponding to acomplement of a nucleic acid sequence found in a transcript or partthereof or molecules with sufficient complementarity to act as antisensemolecules. Such molecules include sequences, without limitation, thatare the complement of those that encode for a protein, fragment thereofor polypeptide, and are the complement of those that correspond totranscribed introns, transcribed 3′ untranslated regions (UTRs), andtranscribed 5′ UTRs.

[0127] Antisense approaches are a way of preventing or reducing genefunction by targeting the genetic material (Mol et al., FEBS Lett.268:427-430 (1990)). The objective of the antisense approach is to use asequence complementary to the target gene to block its expression andcreate a mutant cell line or organism in which the level of a singlechosen protein is selectively reduced or abolished. The site ofinactivation and its developmental effect can be manipulated by thechoice of promoter for antisense genes or by the timing of externalapplication or microinjection. Antisense can manipulate its specificityby selecting either unique regions of the target gene or regions whereit shares homology to other related genes (Hiatt et al., In: GeneticEngineering, Setlow (ed.), Vol. 11, New York: Plenum 49-63 (1989)).

[0128] Antisense RNA techniques involve introduction of RNA that iscomplementary to the target mRNA into cells, which results in specificRNA:RNA duplexes being formed by base pairing between the antisensesubstrate and the target mRNA (Green et al., Annu. Rev. Biochem.55:569-597 (1986)). Under one embodiment, the process involves theintroduction and expression of an antisense gene sequence. Such asequence is one in which part or all of the normal gene sequences areplaced under a promoter in inverted orientation so that the ‘wrong’ orcomplementary strand is transcribed into a noncoding antisense RNA thathybridizes with the target mRNA and interferes with its expression(Takayama and Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155-184 (1990)).An antisense vector is constructed by standard procedures and introducedinto cells by transformation, transfection, electroporation,microinjection, infection, etc. The type of transformation and choice ofvector will determine whether expression is transient or stable. Thepromoter used for the antisense gene may influence the level, timing,tissue, specificity, or inducibility of the antisense inhibition.

[0129] c. Cosuppression or Sense Suppression

[0130] Sense suppression molecules which can be used for gene silencinginclude any molecules that comprise nucleic acid sequences correspondingto a nucleic acid sequence found in a transcript or part thereof ormolecules with sufficient complementarity to act as sense molecules.Such molecules include sequences, without limitation, that encode for aprotein, fragment thereof or polypeptide, and those that correspond totranscribed introns, transcribed 3′ untranslated regions (UTRs), andtranscribed 5′ UTRs

[0131] Cosuppression is the reduction in expression levels, usually atthe level of RNA, of a particular endogenous gene or gene family by theexpression of a homologous sense construct that is capable oftranscribing mRNA of the same strandedness as the transcript of theendogenous gene (Napoli et al., Plant Cell 2:279-289 (1990); van derKrol et al., Plant Cell 2:291-299 (1990)). Cosuppression may result fromstable transformation with a single copy nucleic acid molecule that ishomologous to a nucleic acid sequence found within the cell (Prolls andMeyer, Plant J. 2:465-475 (1992)) or with multiple copies of a nucleicacid molecule that is homologous to a nucleic acid sequence found withinthe cell (Mittlesten et al., Mol. Gen. Genet. 244:325-330 (1994)).Genes, even though different, linked to homologous promoters may resultin the cosuppression of the linked genes (Vaucheret, C. R. Acad. Sci.III316:1471-1483 (1993); Flavell, Proc. Natl. Acad. Sci. (U.S.A.)91:3490-3496 (1994); van Blokland et al., Plant J. 6:861-877 (1994);Jorgensen, Trends Biotechnol. 8:340-344 (1990); Meins and Kunz, In: GeneInactivation and Homologous Recombination in Plants, Paszkowski (ed.),pp. 335-348, Kluwer Academic, Netherlands (1994)).

[0132] iii. Suppression or Expression Nucleic Acid Molecules

[0133] In one aspect of the present invention, the present inventionprovides a nucleic acid molecule which can encode for two, three, four,five, or more than five proteins, fragments thereof, polypeptides, orpeptides operably linked to a single promoter sequence.

[0134] In another aspect of the present invention, the present inventionprovides a nucleic acid molecule which, when introduced into a cell ororganism, is capable of effectively eliminating, substantially reducing,at least partially reducing or reducing the level of two, three, four,five, or more than five mRNA transcripts or proteins encoded by a gene,operably linked to a single promoter sequence.

[0135] C. Other Components of Construct/Vector

[0136] Constructs or vectors may also include, within the region ofinterest, a nucleic acid sequence that acts, in whole or in part, toterminate transcription of that region. A number of such sequences havebeen isolated, including the Tr7 3′ sequence and the NOS 3′ sequence(Ingelbrecht et al., Plant Cell 1:671-680 (1989); Bevan et al., NucleicAcids Res. 11:369-385 (1983)). Regulatory transcript termination regionscan be provided in plant expression constructs of the present inventionas well. Transcript termination regions can be provided by the DNAsequence encoding the gene of interest or a convenient transcriptiontermination region derived from a different gene source, for example,the transcript termination region that is naturally associated with thetranscript initiation region. The skilled artisan will recognize thatany convenient transcript termination region that is capable ofterminating transcription in a plant cell can be employed in theconstructs of the present invention.

[0137] A vector or construct may also include regulatory elements.Examples of such include the Adh intron 1 (Callis et al., Genes andDevelop. 1:1183-1200 (1987)), the sucrose synthase intron (Vasil et al.,Plant Physiol. 91:1575-1579 (1989)) and the TMV omega element (Gallie etal., Plant Cell 1:301-311 (1989)). These and other regulatory elementsmay be included when appropriate.

[0138] A vector or construct may also include a selectable marker.Selectable markers may also be used to select for plants or plant cellsthat contain the exogenous genetic material. Examples of such include,but are not limited to: a neo gene (Potrykus et al., Mol. Gen. Genet.199:183-188 (1985)), which codes for kanamycin resistance and can beselected for using kanamycin, RptII, G418, hpt; a bar gene which codesfor bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al.,Bio/Technology 6:915-922 (1988); Reynaerts et al., Selectable andScreenable Markers, In Gelvin and Schilperoort, Plant Molecular BiologyManual, Kluwer, Dordrecht (1988)); aadA (Scofield et al., Mol. Gen.Genet. 244(2):189-96 (1994)), which encodes glyphosate resistance; anitrilase gene which confers resistance to bromoxynil (Stalker et al.,J. Biol. Chem. 263:6310-6314 (1988)); a mutant acetolactate synthasegene (ALS) which confers imidazolinone or sulphonylurea resistance(European Patent Application 154,204 (Sep. 11, 1985)); ALS (D'Halluin etal., Bio/Technology 10: 309-314 (1992)); and a methotrexate resistantDHFR gene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)).

[0139] A vector or construct may also include a screenable marker.Screenable markers may be used to monitor expression. Exemplaryscreenable markers include: a β-glucuronidase or uidA gene (GUS) whichencodes an enzyme for which various chromogenic substrates are known(Jefferson, Plant Mol. Biol, Rep. 5:387-405 (1987); Jefferson et al.,EMBO J. 6:3901-3907 (1987)); an R-locus gene, which encodes a productthat regulates the production of anthocyanin pigments (red color) inplant tissues (Dellaporta et al., Stadler Symposium 11:263-282 (1988));a β-lactamase gene (Sutcliffe et al., Proc. Natl. Acad. Sci. USA75:3737-3741 (1978)), a gene which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a luciferase gene (Ow et al., Science 234:856-859(1986)); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA80:1101-1105 (1983)) which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikatu et al.,Bio/Technology 8:241-242 (1990)); a tyrosinase gene (Katz et al., J.Gen. Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses tomelanin; and an α-galactosidase gene, which encodes an enzyme which willturn a chromogenic α-galactose substrate.

[0140] Included within the terms “selectable or screenable marker genes”are also genes that encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers that encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes that canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins that are detectable,(e.g., by ELISA), small active enzymes that are detectable inextracellular solution (e.g., α-amylase, β-lactamase, phosphinothricintransferase), or proteins that are inserted or trapped in the cell wall(such as proteins that include a leader sequence such as that found inthe expression unit of extension or tobacco PR-S). Other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art.

[0141] Transgenic Plants, Parts Thereof and Plant Cells

[0142] Exogenous genetic material may be transferred into a plant celland the plant cell can be regenerated into a whole, fertile or sterileplant or plant part. Exogenous genetic material is any genetic material,whether naturally occurring or otherwise, from any source that iscapable of being inserted into any organism. Such exogenous geneticmaterial includes, without limitation, nucleic acid molecules andconstructs that comprise a nucleic acid sequence of the presentinvention, as set forth within.

[0143] In a preferred aspect, a plant cell or plant of the presentinvention includes a nucleic acid molecule comprising a first and secondnucleic acid sequence, where the first nucleic acid sequence which, whenit is expressed, is capable of at least partially enhancing, increasing,enhancing, or substantially enhancing the level of an mRNA transcript orprotein and where the second nucleic acid sequence exhibits sufficienthomology to one or more plant genes such that when it is expressed, itis capable of effectively eliminating, substantially reducing, or atleast partially reducing the level of an mRNA transcript or proteinencoded by the gene from which it was derived or any gene which hashomology to that gene.

[0144] It is understood that any methodology that will suppress theexpression of a gene can be used.

[0145] In an aspect of the present invention, a plant cell or plant ofthe present invention includes a nucleic acid molecule that comprises anucleic acid sequence which is capable of increasing the protein,transcript or both encoded by a GMT gene and at the same timeselectively reducing the protein, transcript or both encoded by a FAD2gene.

[0146] In a preferred aspect, a plant cell or plant of the presentinvention includes a nucleic acid molecule that comprises a firstnucleic acid segment and a second nucleic acid segment, where the firstnucleic acid segment, the second nucleic acid segment, or both, arecapable of altering seed oil composition. In a more preferred aspect,the first nucleic acid sequence, when it is expressed, is capable ofincreasing the level of alpha-tocopherol, and the second nucleic acidsegment exhibits sufficient homology to complements of one or more plantgenes such that when it is expressed, it is capable of increasing thelevel of oleic acid or oil content, or both, the first nucleic acidsequence and the second nucleic acid sequence being operably linked to asingle promoter sequence.

[0147] Genetic material may be introduced into any species, for example,without limitation monocotyledons or dicotyledons, including, but notlimited to alfalfa, apple, Arabidopsis, banana, barley, Brassicacampestris, canola, castor bean, chrysanthemum, coffee, cotton,cottonseed, corn, crambe, cranberry, cucumber, dendrobium, dioscorea,eucalyptus, fescue, flax, gladiolus, liliacea, linseed, millet,muskmelon, mustard, oat, oil palm, oilseed rape, papaya, peanut,perennial, Phaseolus, potato, rapeseed, rice, rye, ryegrass, safflower,sesame, sorghum, soybean, sugarbeet, sugarcane, sunflower, tobacco,tomato, turfgrass, and wheat (Christou, INO: Particle Bombardment forGenetic Engineering of Plants, Biotechnology Intelligence Unit. AcademicPress, San Diego, Calif. (1996)), with alfalfa, Arabidopsis, Brassicacampestris, canola, castor bean, corn, cotton, cottonseed, crambe, flax,linseed, mustard, oil palm, oilseed rape, peanut, potato, rapeseed,safflower, sesame, soybean, sunflower, tobacco, tomato, and wheatpreferred, and Brassica campestris, canola, corn, oil palm, oilseedrape, peanut, rapeseed, safflower, soybean, and sunflower morepreferred. In a more preferred aspect, genetic material is transferredinto canola. In another more preferred aspect, genetic material istransferred into oilseed rape. In another particularly preferredembodiment, genetic material is transferred into soybean or corn.

[0148] Genetic material may also be introduced into a suitable cell suchas a plant cell. The cell may be present in a multi-cellularenvironment. In an aspect of the present invention, the multicellularenvironment may be in a transformed plant.

[0149] Genetic material may also be introduced into a cell or organismsuch as a mammalian cell, mammal, fish cell, fish, bird cell, bird,algae cell, algae, fungal cell, fungi, or bacterial cell. Preferred hostand transformants include: fungal cells such as Aspergillus, yeasts,mammals, particularly bovine and porcine, insects, bacteria, and algae.Particularly preferred bacteria are Agrobacteruim tumefaciens and E.coli.

[0150] The levels of products such as transcripts or proteins may beincreased or decreased or both throughout an organism such as a plant orlocalized in one or more specific organs or tissues of the organism. Forexample the levels of products may be increased or decreased in one ormore of the tissues and organs of a plant including without limitation:roots, tubers, stems, leaves, stalks, fruit, berries, nuts, bark, pods,seeds and flowers. A preferred organ is a seed.

[0151] In an aspect of the invention, after transformation of a plant orother organism with a nucleic acid of the present invention, the levelof one or more agents is at least partially enhanced, increased,enhanced, or substantially enhanced, while a second agent issimultaneously expressed, coexpressed, or coordinately expressed withthe first agent.

[0152] In another aspect, after transformation of a plant or otherorganism with a nucleic acid of the present invention, the level of oneor more agents is at least partially enhanced, increased, enhanced, orsubstantially enhanced, while a second agent is simultaneouslyexpressed, coexpressed, or coordinately expressed, and the simultaneousexpression, coexpression or coordinate expression of the second agentresults in a reduction, preferably at least a partial reduction,substantial reduction or effective elimination of another agent.

[0153] In another aspect, after transformation of a plant or otherorganism with a nucleic acid of the present invention, the level of oneor more agents is at least partially enhanced, increased, enhanced, orsubstantially enhanced, while a second agent is simultaneouslyexpressed, coexpressed, or coordinately expressed with two or greaterthan two agents.

[0154] In another aspect, after transformation of a plant or otherorganism with a nucleic acid of the present invention, the level of oneor more agents is at least partially enhanced, increased, enhanced, orsubstantially enhanced, while a second agent is simultaneouslyexpressed, coexpressed, or coordinately expressed with three or greaterthan three agents.

[0155] In another aspect, after transformation of a plant or otherorganism with a nucleic acid of the present invention, the level of oneor more agents is at least partially enhanced, increased, orsubstantially enhanced while additional agents are simultaneouslyexpressed, coexpressed or coordinately expressed with the first agentand the simultaneous expression, coexpression or coordinated expressionof the additional agents, preferably two or more, three or more, four ormore, or five or more agents, result in at least partial reduction,substantial reduction or an effective elimination of more than oneagent, preferably two or more, three or more, four or more, or five ormore agents.

[0156] In an aspect, after transformation of a plant or other organismwith a nucleic acid of the present invention, one or more agents is atleast partially enhanced, increased, enhanced, or substantially enhancedwhile another agent or agents is simultaneously expressed, coexpressed,or coordinately expressed and such expression results in at least apartial reduction, a substantial reduction, or effective elimination ofan agent or agents.

[0157] When levels of an agent are compared, such a comparison ispreferably carried out between organisms with a similar geneticbackground. In a preferred aspect, a similar genetic background is abackground where the organisms being compared share 50% or greater oftheir nuclear genetic material. In a more preferred aspect a similargenetic background is a background where the organisms being comparedshare 75% or greater, even more preferably 90% or greater of theirnuclear genetic material. In another even more preferable aspect, asimilar genetic background is a background where the organisms beingcompared are plants, and the plants are isogenic except for any geneticmaterial originally introduced using plant transformation techniques.

[0158] In a preferred aspect, the capability of a nucleic acid sequenceto partially enhance, enhance or substantially enhance the level of anagent is carried out by a comparison of levels of mRNA transcripts. In apreferred aspect, the capability of a nucleic acid sequence to partiallyenhance, enhance, or substantially enhance the level of a gene relativeto another gene is carried out by a comparison of levels of proteins,fragments thereof or polypeptides encoded by the genes. In a preferredaspect, the capability of a nucleic acid sequence to reduce the level ofa gene relative to another gene is carried out by a comparison of levelsof mRNA transcripts. In a preferred aspect, the capability of a nucleicacid sequence to reduce the level of a gene relative to another gene iscarried out by a comparison of levels of proteins, fragments thereof orpolypeptides encoded by the genes. As used herein, mRNA transcriptsinclude processed and non-processed mRNA transcripts. As used herein,proteins, fragments thereof or polypeptides include proteins, fragmentsthereof or polypeptides with or without any post-translationalmodification. In another preferred aspect, the capability of a nucleicacid molecule to increase the level of a gene relative to another geneis carried out by a comparison of phenotype. In a preferred aspect, thecomparison of phenotype is a comparison of alpha-tocopherol content. Ina preferred aspect, the comparison of phenotype is a comparison of fattyacid composition. In a preferred aspect, the comparison of phenotype isa comparison of total oil level.

[0159] Methods of Introducing Nucleic Acid Molecules into Plants orOrganisms

[0160] There are many methods for introducing nucleic acid moleculesinto plant cells. Suitable methods are believed to include virtually anymethod by which nucleic acid molecules may be introduced into a cell,such as by Agrobacterium infection or direct delivery of nucleic acidmolecules such as, for example, by transfection, injection, projection,PEG-mediated transformation, by electroporation or by acceleration ofDNA coated particles, and the like. (Potrykus, Ann. Rev. Plant Physiol.Plant Mol. Biol. 42:205-225 (1991); Vasil, Plant Mol. Biol. 25:925-937(1994)). For example, electroporation has been used to transform cornprotoplasts (Fromm et al., Nature 312:791-793 (1986)).

[0161] Nucleic acids can also be introduced into an organism via methodsincluding, but not limited to, conjugation, endocytosis, andphagocytosis. Furthermore, the nucleic acid can be introduced into acell or organism derived from a plant, plant cell, algae, algae cell,fungus, fungal cell, bacterial cell, mammalian cell, fish cell, or birdcell. Particularly preferred microorganisms are E. coli andAgrobacterium species.

[0162] Technology for introduction of DNA into cells is well known tothose of skill in the art. Four general methods for delivering a geneinto cells have been described: (1) chemical methods (Graham and van derEb, Virology 54:536-539 (1973)); (2) physical methods such asmicroinjection (Capecchi, Cell 22:479-488 (1980)), electroporation (Wongand Neumann, Biochem. Biophys. Res. Commun. 107:584-587 (1982); Fromm etal., Proc. Natl. Acad. Sci. USA 82:5824-5828 (1985); U.S. Pat. No.5,384,253); the gene gun (Johnston and Tang, Methods Cell Biol.43:353-365 (1994)); and vacuum infiltration (Bechtold et al., C.R. Acad.Sci. Paris, Life Sci. 316:1194-1199. (1993)); (3) viral vectors (Clapp,Clin. Perinatol. 20:155-168 (1993); Lu et al., J. Exp. Med.178:2089-2096 (1993); Eglitis and Anderson, Biotechniques 6:608-614(1988)); and (4) receptor-mediated mechanisms (Curiel et al., Hum. Gen.Ther. 3:147-154 (1992); Wagner et al., Proc. Natl. Acad. Sci. USA89:6099-6103 (1992)).

[0163] Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules into plant cells ismicroprojectile bombardment. This method has been reviewed by Yang andChristou (eds.), Particle Bombardment Technology for Gene Transfer,Oxford Press, Oxford, England (1994). Non-biological particles(microprojectiles) may be coated with nucleic acid molecules anddelivered into cells by a propelling force. Exemplary particles includethose comprised of tungsten, gold, platinum and the like.

[0164] A particular advantage of microprojectile bombardment, inaddition to it being an effective way of reproducibly transformingmonocots, is that neither the isolation of protoplasts (Cristou et al.,Plant Physiol. 87:671-674 (1988)) nor the susceptibility toAgrobacterium infection is required. An illustrative embodiment of amethod for delivering DNA into corn cells by acceleration is abiolistics α-particle delivery system, which can be used to propelparticles coated with DNA through a screen, such as a stainless steel orNytex screen, onto a filter surface covered with corn cells cultured insuspension. Gordon-Kamm et al., describes the basic procedure forcoating tungsten particles with DNA (Gordon-Kamm et al., Plant Cell2:603-618 (1990)). The screen disperses the tungsten nucleic acidparticles so that they are not delivered to the recipient cells in largeaggregates. A particle delivery system suitable for use with theinvention is the helium acceleration PDS-1000/He gun, which is availablefrom Bio-Rad Laboratories (Bio-Rad, Hercules, Calif.) (Sanford et al.,Technique 3:3-16 (1991)).

[0165] For the bombardment, cells in suspension may be concentrated onfilters. Filters containing the cells to be bombarded are positioned atan appropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

[0166] Alternatively, immature embryos or other target cells may bearranged on solid culture medium. The cells to be bombarded arepositioned at an appropriate distance below the microprojectile stoppingplate. If desired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain 1000 or more loci of cellstransiently expressing a marker gene. The number of cells in a focusthat express the exogenous gene product 48 hours post-bombardment oftenranges from one to ten, and average one to three.

[0167] In bombardment transformation, one may optimize thepre-bombardment culturing conditions and the bombardment parameters toyield the maximum numbers of stable transformants. Both the physical andbiological parameters for bombardment are important in this technology.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmids. It is believed that pre-bombardment manipulationsare especially important for successful transformation of immatureembryos.

[0168] Accordingly, it is contemplated that one may wish to adjustvarious aspects of the bombardment parameters in small-scale studies tofully optimize the conditions. One may particularly wish to adjustphysical parameters such as gap distance, flight distance, tissuedistance and helium pressure. One may also minimize the trauma reductionfactors by modifying conditions that influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

[0169] Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example the methods described by Fraley etal., Bio/Technology 3:629-635 (1985) and Rogers et al., Methods Enzymol.153:253-277 (1987). Further, the integration of the Ti-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., Mol. Gen. Genet. 205:34 (1986)).

[0170] Modern Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described in Klee et al., in Plant DNA InfectiousAgents, Hohn and Schell (eds.), Springer-Verlag, New York, pp. 179-203(1985). Moreover, technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide-coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes (Rogerset al., Methods Enzymol. 153:253-277 (1987)). In addition, Agrobacteriumcontaining both armed and disarmed Ti genes can be used for thetransformations. In those plant strains where Agrobacterium-mediatedtransformation is efficient, it is the method of choice because of thefacile and defined nature of the gene transfer.

[0171] A transgenic plant formed using Agrobacterium transformationmethods typically contains a single gene on one chromosome. Suchtransgenic plants can be referred to as being heterozygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant, a transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants produced for the gene of interest.

[0172] It is also to be understood that two different transgenic plantscan also be mated to produce offspring that contain two independentlysegregating, exogenous constructs. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes thatencode a polypeptide of interest. Backcrossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation.

[0173] Transformation of plant protoplasts can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation and combinations of these treatments (See, e.g.,Potrykus et al., Mol. Gen. Genet. 205:193-200 (1986); Lorz et al., Mol.Gen. Genet. 199:178 (1985); Fromm et al., Nature 319:791 (1986);Uchimiya et al., Mol. Gen. Genet. 204:204 (1986); Marcotte et al.,Nature 335:454-457 (1988)). Application of these systems to differentplant strains depends upon the ability to regenerate that particularplant strain from protoplasts. Illustrative methods for the regenerationof cereals from protoplasts are described (Fujimura et al., Plant TissueCulture Letters 2:74 (1985); Toriyama et al., Theor. Appl. Genet. 205:34(1986); Yamada et al., Plant Cell Rep. 4:85 (1986); Abdullah et al.,Biotechnology 4:1087 (1986)).

[0174] To transform plant strains that cannot be successfullyregenerated from protoplasts, other ways to introduce DNA into intactcells or tissues can be utilized. For example, regeneration of cerealsfrom immature embryos or explants can be effected as described (Vasil,Bio/Technology 6:397 (1988)). In addition, “particle gun” orhigh-velocity microprojectile technology can be utilized (Vasil et al.,Bio/Technology 10:667 (1992)). Using the latter technology, DNA iscarried through the cell wall and into the cytoplasm on the surface ofsmall metal particles as described (Klein et al., Nature 328:70 (1987);Klein et al., Proc. Natl. Acad. Sci. USA 85:8502-8505 (1988); McCabe etal., Bio/Technology 6:923 (1988)). The metal particles penetrate throughseveral layers of cells and thus allow the transformation of cellswithin tissue explants.

[0175] Methods for transforming dicots, primarily by use ofAgrobacterium tumefaciens and obtaining transgenic plants have beenpublished for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135;U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834; U.S. Pat.No. 5,416,011; McCabe et al., Biotechnology 6:923 (1988); Christou etal., Plant Physiol. 87:671-674 (1988)); Brassica (U.S. Pat. No.5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653-657 (1996),McKently et al., Plant Cell Rep. 14:699-703 (1995)); papaya; pea (Grantet al., Plant Cell Rep. 15:254-258 (1995)); and Arabidopsis thaliana(Bechtold et al., C.R. Acad. Sci. Paris, Life Sci. 316:1194-1199(1993)). The latter method for transforming Arabidopsis thaliana iscommonly called “dipping” or vacuum infiltration or germplasmtransformation.

[0176] Transformation of monocotyledons using electroporation, particlebombardment and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354 (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); corn (Rhodes et al., Science 240:204(1988); Gordon-Kamm et al., Plant Cell 2:603-618 (1990); Fromm et al.,Bio/Technology 8:833 (1990); Koziel et al., Bio/Technology 11:194(1993); Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somerset al., Bio/Technology 10:1589 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., Theor Appl. Genet.205:34 (1986); Part et al., Plant Mol. Biol. 32:1135-1148 (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al., Plant Cell Rep.7:379 (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., Bio/Technology 10:691 (1992)) and wheat (Vasilet al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

[0177] Assays for gene expression based on the transient expression ofcloned nucleic acid constructs have been developed by introducing thenucleic acid molecules into plant cells by polyethylene glycol (PEG)treatment, electroporation, or particle bombardment (Marcotte et al.,Nature 335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)). Transientexpression systems may be used to functionally dissect gene constructs(see generally, Maliga et al., Methods in Plant Molecular Biology, ColdSpring Harbor Press (1995)).

[0178] Any of the nucleic acid molecules of the invention may beintroduced into a plant cell in a permanent or transient manner. Anucleic acid molecule of the present invention may be stably integratedinto a nuclear, chloroplast or mitochondrial genome, preferably into thenuclear genome.

[0179] Other methods of cell or organism transformation can also be usedand include but are not limited to introduction of DNA into plants bydirect DNA transfer into pollen (Hess et al., Intern Rev. Cytol. 107:367(1987); Luo et al., Plant Mol Biol. Reporter 6:165 (1988)), by directinjection of DNA into reproductive organs of a plant (Pena et al.,Nature 325:274 (1987)), by direct microinjection of DNA into protoplasts(Crossway et al., Mol. Gen. Genet. 202: 179-185 (1986)), or by directinjection of DNA into the cells of immature embryos followed by therehydration of desiccated embryos (Neuhaus et al., Theor. Appl. Genet.75:30 (1987)). See also EP 0 238 023; Yelton et al., Proc. Natl. Acad.Sci. (U.S.A.), 81:1470-1474 (1984); Malardier et al., Gene, 78:147-156(1989); Becker and Guarente, In: Abelson and Simon (eds.), Guide toYeast Genetics and Molecular Biology, Method Enzymol., Vol. 194, pp.182-187, Academic Press, Inc., New York; Ito et al., J. Bacteriology,153:163 (1983); Hinnen et al., Proc. Natl. Acad. Sci. (U.S.A.), 75:1920(1978); and Bennett and LaSure (eds.), More Gene Manipualtionins infungi, Academic Press, CA (1991).

[0180] The regeneration, development and cultivation of plants fromsingle plant protoplast transformants or from various transformedexplants are well known in the art (Weissbach and Weissbach, In Methodsfor Plant Molecular Biology, Academic Press, San Diego, Calif., (1988)).This regeneration and growth process typically includes the steps ofselection of transformed cells and culturing those individualized cellsthrough the usual stages of embryonic development and through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil.

[0181] The development or regeneration of plants containing a foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of theinvention containing a desired polypeptide is cultivated using methodswell known to one skilled in the art.

[0182] There are a variety of methods for the regeneration of plantsfrom plant tissue. The particular method of regeneration will depend onthe starting plant tissue and the particular plant species to beregenerated.

[0183] The present invention also provides for the generation of partsof the plants, particularly reproductive or storage parts. Plant parts,without limitation, include seeds, endosperm, ovule, pollen, roots,tubers, stems, leaves, stalks, fruit, berries, nuts, bark, pods, andflowers. In a particularly preferred embodiment of the presentinvention, the plant part is a seed.

[0184] Any of the plants or parts thereof of the present invention maybe processed to produce a feed, meal, protein, or oil preparation. Aparticularly preferred plant part for this purpose is a seed. In apreferred embodiment, the feed, meal, protein or oil preparation isdesigned for livestock animals or humans, or both. Methods to producefeed, meal, protein and oil preparations are known in the art. See, forexample, U.S. Pat. Nos. 4,957,748, 5,100,679, 5,219,596, 5,936,069,6,005,076, 6,146,669, and 6,156,227. In a preferred embodiment, theprotein preparation is a high protein preparation. Such a high proteinpreparation preferably has a protein content of greater than 5% w/v,more preferably 10% w/v, and even more preferably 15% w/v. In apreferred oil preparation, the oil preparation is a high oil preparationwith an oil content derived from a plant or part thereof of the presentinvention of greater than 5% w/v, more preferably 10% w/v, and even morepreferably 15% w/v. In a preferred embodiment, the oil preparation is aliquid. In a preferred embodiment, the oil preparation is of a volumegreater than 1, 5, 10 or 50 liters. The present invention provides foroil produced from plants of the present invention or generated by amethod of the present invention. Such oil may exhibit enhanced oxidativestability. Also, such oil may be a minor or major component of anyresultant product. Moreover, such oil may be blended with other oils. Ina preferred embodiment, the oil produced from plants of the presentinvention or generated by a method of the present invention constitutesgreater than 0.5%, 1%, 5%, 10%, 25%, 50%, 75% or 90% by volume or weightof the oil component of any composition. In another embodiment, the oilpreparation may be blended and can constitute greater than 10%, 25%,35%, 50% or 75% of the blend by volume. Oil produced from a plant of thepresent invention can be admixed with one or more organic solvents orpetroleum distillates.

[0185] Plants of the present invention can be part of or generated froma breeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). Selected, non-limiting approaches, for breedingthe plants of the present invention are set forth below. A breedingprogram can be enhanced using marker-assisted selection of the progenyof any cross. It is further understood that any commercial andnon-commercial cultivars can be utilized in a breeding program. Factorssuch as, for example, emergence vigor, vegetative vigor, stresstolerance, disease resistance, branching, flowering, seed set, seedsize, seed density, standability, and threshability will generallydictate the choice.

[0186] For highly heritable traits, a choice of superior individualplants evaluated at a single location will be effective, whereas fortraits with low heritability, selection should be based on mean valuesobtained from replicated evaluations of families of related plants.Popular selection methods commonly include pedigree selection, modifiedpedigree selection, mass selection, and recurrent selection. In apreferred embodiment, a backcross or recurrent breeding program isundertaken.

[0187] The complexity of inheritance influences choice of the breedingmethod. Backcross breeding can be used to transfer one or a fewfavorable genes for a highly heritable trait into a desirable cultivar.This approach has been used extensively for breeding disease-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

[0188] Breeding lines can be tested and compared to appropriatestandards in environments representative of the commercial targetarea(s) for two or more generations. The best lines are candidates fornew commercial cultivars; those still deficient in traits may be used asparents to produce new populations for further selection.

[0189] One method of identifying a superior plant is to observe itsperformance relative to other experimental plants and to a widely grownstandard cultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of its genetic worth. Abreeder can select and cross two or more parental lines, followed byrepeated selfing and selection, producing many new genetic combinations.

[0190] The development of new cultivars requires the development andselection of varieties, the crossing of these varieties and theselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Hybrids are selected for certain single gene traitssuch as pod color, flower color, seed yield, pubescence color, orherbicide resistance, which indicate that the seed is truly a hybrid.Additional data on parental lines, as well as the phenotype of thehybrid, influence the breeder's decision whether to continue with thespecific hybrid cross.

[0191] Pedigree breeding and recurrent selection breeding methods can beused to develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

[0192] Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. A F₂ population is produced byselfing one or several F₁'s. Selection of the best individuals from thebest families is carried out. Replicated testing of families can beginin the F₄ generation to improve the effectiveness of selection fortraits with low heritability. At an advanced stage of inbreeding (i.e.,F₆ and F₇), the best lines or mixtures of phenotypically similar linesare tested for potential release as new cultivars.

[0193] Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting parent is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

[0194] The single-seed descent procedure in the strict sense refers toplanting a segregating population, harvesting a sample of one seed perplant, and using the one-seed sample to plant the next generation. Whenthe population has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

[0195] In a multiple-seed procedure, breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

[0196] The multiple-seed procedure has been used to save labor atharvest. It is considerably faster to thresh pods with a machine than toremove one seed from each by hand for the single-seed procedure. Themultiple-seed procedure also makes it possible to plant the same numberof seed of a population each generation of inbreeding.

[0197] Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g. Fehr, Principles of Cultivar Development Vol. 1, pp. 2-3(1987)).

[0198] A transgenic plant of the present invention may also bereproduced using apomixis. Apomixis is a genetically controlled methodof reproduction in plants where the embryo is formed without union of anegg and a sperm. There are three basic types of apomicticreproduction: 1) apospory where the embryo develops from a chromosomallyunreduced egg in an embryo sac derived from the nucleus, 2) diplosporywhere the embryo develops from an unreduced egg in an embryo sac derivedfrom the megaspore mother cell, and 3) adventitious embryony where theembryo develops directly from a somatic cell. In most forms of apomixis,pseudogamy or fertilization of the polar nuclei to produce endosperm isnecessary for seed viability. In apospory, a nurse cultivar can be usedas a pollen source for endosperm formation in seeds. The nurse cultivardoes not affect the genetics of the aposporous apomictic cultivar sincethe unreduced egg of the cultivar develops parthenogenetically, butmakes possible endosperm production. Apomixis is economically important,especially in transgenic plants, because it causes any genotype, nomatter how heterozygous, to breed true. Thus, with apomicticreproduction, heterozygous transgenic plants can maintain their geneticfidelity throughout repeated life cycles. Methods for the production ofapomictic plants are known in the art. See, e.g., U.S. Pat. No.5,811,636.

[0199] The following examples are illustrative and not intended to belimiting in any way.

EXAMPLE 1

[0200] This example illustrates constructs which were prepared todemonstrate the practice of this invention.

[0201] With reference to FIG. 1 there is shown schematically theelements of a DNA construct comprising in series

[0202] (a) DNA of a napin promoter,

[0203] (b) DNA coding for gamma methyl transferenase (GMT) isolated fromGossypium hirsutium (cotton),

[0204] (c) sense oriented DNA of the 3′ UTR of Arabidopsis thalianafad2,

[0205] (d) DNA of an intron in the Arabidopsis thaliana fad2 with splicesites removed,

[0206] (e) the complement of the (c) element, i.e. the antisenseoriented DNA of the 3′UTR of Arabidopsis thaliana fad2, and

[0207] (f) DNA of a napin 3′ terminator.

[0208] The construct was inserted together with a BAR marker elementinto a vector between TI borders from Agrobacterium tumefaciens. Withreference to SEQ ID NO: 5 the pertinent DNA elements of a vector, whichwas designated pMON75565, are described in Table 1. TABLE 1 Elements ofvector pMON75565 Bases description of DNA segment  1-285 Agrobacteriumtumefaciens right border  520-2282 napin promoter 2344-3381 Gossypiumhirsutium gmt 3425-3470 napin 3′ transcription terminator 3545-3678 fad23′ UTR in sense orientation 3687-4818 fad2 intron 4823-4947 fad2 3′ UTRin antisense orientation 4985-6199 napin 3′ transcription terminator6381-6780 CaMV 35S promoter 6781-7328 BAR marker gene 7333-7590 NOStranscription terminator 7597-8179 Agrobacterium tumefaciens left border

[0209] With reference to FIG. 2 there is shown schematically theelements of a DNA construct comprising in series

[0210] (a) DNA of a napin promoter,

[0211] (b) DNA coding for gamma methyl transferenase (GMT) isolated fromGossypium hirsutium (cotton),

[0212] (c) DNA of an intron in the Arabidopsis thaliana fad2 with splicesites removed, and

[0213] (d) DNA of a napin 3′ terminator.

[0214] The construct was inserted together with a BAR marker elementinto a vector between TI borders from Agrobacterium tumefaciens. Withreference to SEQ ID NO: 6 the pertinent DNA elements of a vector, whichwas designated pMON75571, are described in Table 2. TABLE 2 Elements ofvector pMON75571 Bases description of DNA segment  1-285 Agrobacteriumtumefaciens right border  520-2282 napin promoter 2344-3381 Gossypiumhirsutium gmt 3396-4515 fad2 intron 4519-5733 napin 3′ transcriptionterminator 5915-6314 CaMV 35S promoter 6315-6862 BAR marker gene6867-7124 NOS transcription terminator 7131-7713 Agrobacteriumtumefaciens left border

[0215] Transformation of Plants with pMON75565 and pMON75571

[0216] Vectors, pMON75565 and pMON75571, are used in Arabidopsisthaliana plant transformation to direct the expression of GMT andinhibit the expression of the fad2 gene. Binary vector constructspMON75565 and pMON75571 are transformed into ABI strain Agrobacteriumcells by the method of Holsters et al., Mol. Gen. Genet. 163:181-187(1978). Transgenic Arabidopsis thaliana plants are obtained byAgrobacterium-mediated transformation as described by Valverkens et al.,Proc. Nat. Acad. Sci. USA 85:5536-5540 (1988), Bent et al., Science265:1856-1860 (1994), and Bechtold et al., C.R. Acad. Sci., LifeSciences 316:1194-1199 (1993). Transgenic plants are selected bysprinkling the transformed R₁ seeds directly onto soil and thenvernalizing them at 4° C. in the absence of light for 4 days. The seedsare then transferred to 21° C., 16 hours light and sprayed with a 1:200dilution of Finale (Basta) herbicide at 7 days and 14 days afterseeding. Transformed plants are grown to maturity and the R₂ seed thatis produced is analyzed for tocopherol content.

[0217]FIG. 3 shows data from the alpha-tocopherol level analysis from R₂seed of transgenic Arabidopsis thaliana plants expressing GMTs frompMON75565 (top) or pMON75571 (bottom) under the control of the napinseed-specific promoter. Table 3 below gives specific tocopherol levelresults (alpha, gamma and delta) for various transformed and controlplant lines. TABLE 3 alpha gamma delta Construct Toco Toco Toco totalToco % alpha Generation Control 7 453 12 472 1.5 R3 9 446 12 467 1.9 R35 440 10 455 1.1 R3 7 460 12 479 1.5 R3 9 460 13 482 1.9 R3 6 443 10 4591.3 R3 6 459 11 476 1.3 R3 8 456 10 474 1.7 R3 6 447 11 464 1.3 R3 7 4369 452 1.5 R3 pMON 67 386 11 464 14.4 R2 75565 320 152 5 477 67.1 R2 304142 6 452 67.3 R2 309 142 5 456 67.8 R2 292 134 4 430 67.9 R2 320 143 5468 68.4 R2 360 145 5 510 70.6 R2 317 121 4 442 71.7 R2 329 124 4 45772.0 R2 336 79 3 418 80.4 R2 369 78 3 450 82.0 R2 392 68 4 464 84.5 R2391 66 4 461 84.8 R2 422 51 2 475 88.8 R2 pMON 10 492 13 515 1.9 R275571 137 350 8 495 27.7 R2 296 166 5 467 63.4 R2 313 136 5 454 68.9 R2364 124 4 492 74.0 R2 354 119 3 476 74.4 R2 371 91 2 464 80.0 R2 381 872 470 81.1 R2 391 52 2 445 87.9 R2 422 55 3 480 87.9 R2 436 54 2 49288.6 R2 410 45 2 457 89.7 R2 449 45 1 495 90.7 R2 439 31 1 471 93.2 R2475 22 1 498 95.4 R2

[0218]FIG. 3 and Table 3 show that the construct increased the level ofalpha-tocopherol in the transformed plant lines compared withnon-transformed plant lines.

[0219] Fatty acid compositions are analyzed using gas chromatographyfrom seed of Arabidopsis lines transformed with constructs pMON75565 andpMON75571. Table 4 provides a summary of fatty acid levels that areobtained using these constructs. As can be seen, the expression thepMON75565 construct results in increased expression of oleic acid(18: 1) and minor decrease in the expression of linoleic acid (18:2) andlinolenic acid (18:3), with virtually no change in the levels of stearicacid (18:0). There are no significant changes in 12:0, 14:0, 16:0, 16:1,20:0, 20:1, 20:2, 22:0, 22:1 and 22:2 fatty acid levels. The results forpMON75571 and pMON75565 differ. Moreover, there is a higher percentageof success using RNAi suppression as compared to sense suppression.

[0220] Table 5 provides a summary of oil levels that are obtained usingthe described constructs. As can be seen, the total levels of protein,carbon, nitrogen and sulfur remain virtually the same when the pMON75565and pMON75571 constructs are used as compared to the control constructs.

[0221]FIG. 4 depicts a graphic presentation of both fatty acid and oillevels that are obtained using the pMON75565 and pMON75571 constructs.Lines AT_G490 and AT_G499 (both obtained using pMON75565) have thehighest oleic acid and exhibit alpha-tocopherol phenotypes and are bothtaken onto the next generation for tocopherol and oleic acid and oilanalysis. Expression of the double-stranded FAD2 RNA sequences result inthe modification of both the fatty acid and the oil compositions.

[0222] In order to confirm the phenotype of the pMON75565 construct, theR₂ plants expressing the pMON75565 construct are self crossed to obtainR₃ plants. Table 6 confirms that the expression of the double-strandedFAD2 RNA sequences by the R₃ plants result in the modification of boththe fatty acid and the oil compositions. Specifically, the levels ofoleic acid are increased as compared to the control construct, and thelevels of linoleic and linolenic acid are slightly decreased. Such aresult is consistent with a down-regulation of FAD2 expression.

[0223] Table 7 and FIG. 5 confirm that the R₃ plants express the GMT RNAsequence, which results in increased levels of alpha-tocopherol, whilethe total levels of tocopherol remain essentially the same.

[0224] These data show that the constructs of the present inventionup-regulate cotton GMT protein and down-regulate the expression of FAD2.Increased expression of GMT results in an increase in alpha-tocopherollevels. (GMT converts gamma-tocopherol to alpha-tocopherol). An oleicacid level increase and linoleic acid level decrease is consistent withdown regulation. TABLE 4 CONSTRUCT STRAIN ID 18:0 18:1 18:2 18:3 Control9979-54-49 2.98 14.09 28.71 18.88 9979-54-50 2.89 14.28 29.51 18.419979-54-51 2.8 14.46 29.28 18.43 9979-54-52 2.75 15.5 29.53 17.579979-54-53 2.78 15.61 29.39 17.63 pMON AT_G485 3.04 22.4 20.82 18.3875565 AT_G486 2.9 18.09 25.88 18.25 AT_G487 2.95 16.39 26.28 19.71AT_G488 2.97 22.53 20.95 18.16 AT_G489 2.8 28.87 18.17 15.53 AT_G490 332.34 15.18 15.05 AT_G492 2.8 18.26 26.68 17.51 AT_G493 2.86 24.25 21.1616.85 AT_G494 3.02 23.36 20.44 18.12 AT_G495 2.9 23.9 21.43 16.88AT_G496 3.02 21.53 22.08 18.59 AT_G497 2.79 27.9 17.46 16.58 AT_G4982.88 19.35 24.42 18.22 AT_G499 3.04 30.19 17.08 15.55 Control 9979-54-592.84 14.86 29.6 17.91 9979-54-60 2.83 14.96 29.41 18.14 9979-54-61 3.0214.97 29.05 18.62 9979-54-62 2.71 14.78 29.6 18.18 9979-54-63 2.95 15.2930.13 17.43 pMON AT_G500 2.84 15.38 28.74 18.39 75571 AT_G501 2.75 16.7329.31 16.88 AT_G502 2.85 15.86 27.86 18.79 AT_G503 2.8 17.18 29.52 16.38AT_G504 2.9 15.29 29.01 18.38 AT_G505 2.93 16.25 28.94 17.59 AT_G5062.86 16.3 29.18 17.23 AT_G507 2.89 16.31 27.88 18.27 AT_G508 2.98 16.4429.93 16.73 AT_G509 2.89 15.77 28.8 17.9 AT_G510 2.84 16.91 29.78 16.44AT_G511 2.79 15.32 27.82 19.05 AT_G512 2.77 17.88 29.68 15.62 AT_G5132.86 16.7 29.52 16.78 AT_G514 2.86 15.84 28.66 18.19

[0225] TABLE 5 CONSTRUCT EVENT GENERATION % OIL % PRO % C % N % S COLORControl 9979-AT00002-54-49 R3 36.4 22.3 53.4 3.7 0.75 0.9819979-AT00002-54-50 R3 35.4 22.7 52.8 3.8 0.86 0.985 9979-AT00002-54-51R3 35.1 23.5 53 3.9 0.88 0.974 9979-AT00002-54-52 R3 37.3 21.5 53.6 3.60.85 0.978 9979-AT00002-54-53 R3 35.4 23.5 53 3.9 1.03 0.968 pMONAT_G485 R2 32 25.2 51.8 4.2 0.89 0.982 75565 AT_G486 R2 36.9 22.6 53.83.8 0.79 0.981 AT_G487 R2 35.7 23.1 53.1 3.8 0.86 0.98 AT_G488 R2 36.922.5 53.9 3.8 0.74 0.979 AT_G489 R2 37.1 22.2 53.9 3.7 0.91 0.984AT_G490 R2 37.2 22 54 3.7 0.86 0.981 AT_G492 R2 36.8 21.7 53.4 3.6 0.890.986 AT_G493 R2 37.2 22.8 53.9 3.8 0.97 0.976 AT_G494 R2 36.8 22.3 53.73.7 0.8 0.975 AT_G495 R2 36.3 21.7 53.5 3.6 0.9 0.999 AT_G496 R2 36.5 2353.6 3.8 0.8 0.984 AT_G497 R2 35.5 23.5 53.2 3.9 0.95 0.983 AT_G498 R237.1 22.9 53.8 3.8 0.91 0.988 AT_G499 R2 36.5 22.4 53.6 3.7 0.83 0.985Control 9979-AT00002-54-59 R3 36.5 22.5 53.7 3.8 0.96 0.9779979-AT00002-54-60 R3 36.3 22.4 53.6 3.7 0.96 0.978 9979-AT00002-54-61R3 35.9 23 53.5 3.8 0.94 0.976 9979-AT00002-54-62 R3 36.3 22.9 53.6 3.81 0.977 9979-AT00002-54-63 R3 36 22.9 53.6 3.8 0.95 0.975 pMON AT_G500R2 37.1 22.5 53.9 3.7 0.94 0.976 75571 AT_G501 R2 36.2 22.9 53.5 3.81.14 0.971 AT_G502 R2 36.3 23.4 53.7 3.9 1.01 0.976 AT_G503 R2 36.2 22.253.6 3.7 1 0.98 AT_G504 R2 37.1 22.1 53.9 3.7 0.96 0.974 AT_G505 R2 37.421.7 54 3.6 0.88 0.983 AT_G506 R2 38 21.3 54.3 3.6 0.95 0.976 AT_G507 R236.5 23.1 53.7 3.8 1.01 0.974 AT_G508 R2 36.9 22.2 53.8 3.7 0.97 0.981AT_G509 R2 36.7 22.3 53.7 3.7 0.99 0.978 AT_G510 R2 36.9 22.2 53.9 3.70.98 0.978 AT_G511 R2 34.8 23.8 53 4 1 0.982 AT_G512 R2 35 23.7 53.2 3.91.15 0.973 AT_G513 R2 36.1 22.6 53.4 3.8 0.99 0.982 AT_G514 R2 37.3 22.354 3.7 0.96 0.976

[0226] TABLE 6 CONSTRUCT STRAIN ID 18:0 18:1 18:2 18:3 pMON AT_G490-22.95 21.4 23.91 17.38 75565 AT_G490-4 2.99 22.46 22.47 17 AT_G490-3 2.8322.78 22.64 17.13 AT_G490-8 2.88 22.82 22.81 16.59 AT_G490-5 3 23.3322.51 16.51 AT_G490-6 2.93 26.1 20.29 16.02 AT_G490-7 3.07 27 19.7215.89 AT_G490-9 2.99 28.59 18.55 15.59 AT_G490-1 2.94 29.9 18.12 14.83AT_G490-10 2.99 31.8 15.49 14.59 AT_G499-9 3.25 26.35 20.47 16.09AT_G499-1 3.12 27.19 17.99 16.59 AT_G499-6 3.13 28.49 20.52 14.81AT_G499-2 3.05 28.86 19.75 14.73 AT_G499-3 3.11 30.21 18.27 14.88AT_G499-5 3.11 30.76 19.83 13.71 AT_G499-10 3.09 32.56 15.77 14.33AT_G499-8 2.91 32.88 16.02 14.46 AT_G499-4 2.86 33.16 16.08 14.17AT_G499-7 3.67 34.04 14.53 11.07 Control 9979-40-92 2.74 15.3 29.0717.16 9979-40-94 2.64 15.9 29.02 17.16 9979-40-95 2.81 15.92 29.03 17.359979-40-88 2.85 16.17 28.87 17.14 9979-40-97 2.79 16.42 28.9 16.589979-40-90 2.56 16.5 29.15 16.45 9979-40-93 2.72 16.65 29.22 16.319979-40-91 2.67 16.84 29.61 16.33 9979-40-96 2.78 16.88 29.07 16.449979-40-89 2.71 16.92 28.88 16.51 9979-40-100 2.67 14.86 28.84 17.599979-40-105 2.81 15.08 28.3 18 9979-40-99 2.78 15.4 28.78 17.719979-40-101 2.73 15.6 28.74 17.44 9979-40-103 2.85 15.67 29.09 17.349979-40-106 2.69 15.83 28.96 17.31 9979-40-102 2.87 15.94 28.45 17.259979-40-107 2.79 16.75 29.16 16.4 9979-40-104 2.82 16.78 28.41 17.039979-40-98 2.89 16.89 27.99 16.94

[0227] TABLE 7 alpha- gamma- delta- Total % alpha- Construct Strain IDToco Toco Toco Toco Toco Generation Control 9979-40-100 5 495 16 516 1R3 9979-40-94 5 469 15 489 1 R3 9979-40-93 6 468 14 488 1 R3 9979-40-1016 461 14 481 1 R3 9979-40-95 6 455 14 475 1 R3 9979-40-91 7 491 17 515 1R3 9979-40-90 7 491 16 514 1 R3 9979-40-96 7 490 15 512 1 R3 9979-40-997 473 16 496 1 R3 9979-40-106 7 471 15 493 1 R3 9979-40-107 7 469 14 4901 R3 9979-40-103 7 458 14 479 1 R3 9979-40-92 7 447 15 469 1 R39979-40-89 8 498 18 524 2 R3 9979-40-88 8 496 16 520 2 R3 9979-40-102 8485 15 508 2 R3 9979-40-97 8 474 16 498 2 R3 9979-40-98 9 462 14 485 2R3 9979-40-104 9 460 15 484 2 R3 9979-40-105 9 453 15 477 2 R3 pMON75565AT_G499-9. 286 161 7 454 63 R3 AT_G490-8. 268 143 8 419 64 R3 AT_G499-5.274 147 7 428 64 R3 AT_G490-4. 291 153 7 451 65 R3 AT_G490-2. 282 143 7432 65 R3 AT_G499-2. 286 145 7 438 65 R3 AT_G499-6. 301 152 7 460 65 R3AT_G490-5. 274 123 8 405 68 R3 AT_G490-3. 285 128 8 421 68 R3 AT_G490-9.312 116 7 435 72 R3 AT_G490-7. 330 85 6 421 78 R3 AT_G490-10. 330 80 6416 79 R3 AT_G499-3. 352 84 6 442 80 R3 AT_G499-1. 344 71 5 420 82 R3AT_G490-1. 368 71 6 445 83 R3 AT_G499-10. 380 56 5 441 86 R3 AT_G499-4.368 55 4 427 86 R3 AT_G499-7. 441 56 4 501 88 R3 AT_G499-8. 423 48 4 47589 R3 AT_G490-6. 367 34 4 405 91 R3

[0228] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1 6 1 1038 DNA Gossypium hirsutum 1 atggctgccg cgttacaatt acaaacacacccttgcttcc atggcacgtg ccaactctca 60 cctccgccac gaccttccgt ttccttcccttcttcctccc gctcgtttcc atctagcaga 120 cgttccctgt ccgcgcatgt gaaggcggcggcgtcgtctt tgtccaccac caccttgcag 180 gaagggatag cggagtttta cgatgagtcgtcggggattt gggaagacat atggggtgac 240 catatgcacc atggatatta cgagccgggttccgatattt cgggttcaga tcatcgtgcc 300 gctcagattc gaatggtcga agaatcgctccgttttgctg gaatatcaga ggacccagca 360 aacaggccca agagaatagt tgatgttgggtgtgggatag gaggcagttc taggtatcta 420 gcaaggaaat atggggcaaa atgccaaggcattactttga gccctgttca agctggaaga 480 gccaatgctc ttgctaatgc tcaaggactagcagaacagg tttgttttga agttgcagat 540 gccttgaacc aaccattccc tgatgaccaatttgatcttg tttggtctat ggaaagcgga 600 gaacacatgc ctgacaaacc caagtttgttaaagagctgg tgcgagtggc agctccagga 660 ggcacaataa tagtagtgac atggtgccatagggatcttg gtccatctga agagtctttg 720 cagccatggg agcaaaagct tttaaacagaatatgtgatg cttactattt accagagtgg 780 tgttctactt ctgattatgt caaattatttcagtccctat ctctccagga tataaaggca 840 ggagactgga ctgagaatgt agcacccttttggccagcag tgatacgttc agcattgaca 900 tggaagggct tcacatcgct gctacgaagtggattaaaaa caataaaagg tgcactggtg 960 atgccattga tgatcgaagg tttccagaaaggggtgataa agtttgccat cattgcttgc 1020 cggaagccag ctgagtag 1038 2 62 DNAArtificial sequence Synthetic Primer 2 ggggacaagt ttgtacaaaa aagcaggctgcggccgcaca atggctgccg cgttacaatt 60 ac 62 3 57 DNA Artificial SequenceSynthetic Primer 3 ggggaccact ttgtacaaga aagctgggtc ctgcaggctactcagctggc ttccggc 57 4 1405 DNA Artificial sequence Synthetic Primer 4cgcccttcgg ccgcgcatga tggtgaagaa attgtcgacc tttctcttgt ctgtttgtct 60tttgttaaag aagctatgct tcgttctaat aatcttattg tccattttgt tgtgttatga 120cattttggct gctcccatgg caggtccgtc gcttctcttc catttcttct cattttcgat 180tttgattctt atttctttcc agtagctcct gctctgtgaa tttctccgct cacgatagat 240ctgcttatac tccttacatt caaccttaga tctggtctcg attctctgtt tctctgtttt 300tttcttttgg tcgagaatct gatgtttgtt tatgttctgt caccattaat aataatgaac 360tctctcattc atacaatgat tagtttctct cgtctacaaa acgatatgtt gcattttcac 420ttttcttctt tttttctaag atgatttgct ttgaccaatt tgtttagatc tttattttat 480tttattttct ggtgggttgg tggaaattga aaaaaaaaaa aaacagcata aattgttatt 540tgttaatgta ttcatttttt ggctatttgt tctgggtaaa aatctgcttc tactattgaa 600tctttcctgg attttttact cctattgggt ttttatagta aaaatacata ataaaaggaa 660aacaaaagtt ttatagattc tcttaaaccc cttacgataa aagttggaat caaaataatt 720caggatcaga tgctctttga ttgattcaga tgcgattaca gttgcatggc aaattttcta 780gatccgtcgt cacattttat tttctgttta aatatctaaa tctgatatat gatgtcgaca 840aattctggtg gcttatacat cacttcaact gttttctttt ggctttgttt gtcaacttgg 900ttttcaatac gatttgtgat ttcgatcgct gaatttttaa tacaagcaaa ctgatgttaa 960ccacaagcaa gagatgtgac ctgccttatt aacatcgtat tacttactac tagtcgtatt 1020ctcaacgcaa tcgtttttgt atttctcaca ttatgccgct tctctactct ttattccttt 1080tggtccacgc attttctatt tgtggcaatc cctttcacaa cctgatttcc cactttggat 1140catttgtctg aagactctct tgaatcgtta ccacttgttt cttgtgcatg ctctgttttt 1200tagaattaat gataaaacta ttccatagtc ttgagttttc agcttgttga ttcttttgct 1260tttggttttc tgcagggtac cgagcagcca aaatgtcaaa acacaacaaa atggacaata 1320agattattaa aacgaagcat agcttcttta acaaaagaca aacagacaag agaaaggtcg 1380acaatttctt caccatcatg ccccg 1405 5 8179 DNA Artificial Sequence Vector 5cgaagctcgg tcccgtgggt gttctgtcgt ctcgttgtac aacgaaatcc attcccattc 60cgcgctcaag atggcttccc ctcggcagtt catcagggct aaatcaatct agccgacttg 120tccggtgaaa tgggctgcac tccaacagaa acaatcaaac aaacatacac agcgacttat 180tcacacgagc tcaaattaca acggtatata tcctgccagt cagcatcatc acaccaaaag 240ttaggcccga atagtttgaa attagaaagc tcgcaattga ggtctgcgcc caatacgcaa 300accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 360ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 420ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 480atttcacaca ggaaacagct atgaccatga ttacgaattg taccgaatta tcactacaat 540gtcggagaga caaggctgcg ccagcatata caaaagggaa atgaagatgg ccttttgatt 600agctgtgtag catcagcagc taatctctgg gctctcatca tggatgctgg aactggattc 660acttctcaag tttatgagtt gtcaccggtc ttcctacaca aggtaataat cagttgaagc 720aattaagaat caatttgatt tgtagtaaac taagaagaac ttaccttatg ttttccccgc 780aggactggat tatggaacaa tgggaaaaga actactatat aagctccata gctggttcag 840ataacgggag ctctttagtt gttatgtcaa aaggttagtg tttagtgaat aataaactta 900taccacaaag tcttcattga cttatttata tacttgttgt gaattgctag gaactactta 960ttctcagcag tcatacaaag tgagtgactc atttccgttc aagtggataa ataagaaatg 1020gaaagaagat tttcatgtaa cctccatgac aactgctggt aatcgttggg gtgtggtaat 1080gtcgaggaac tctggcttct ctgatcaggt aggtttttgt ctcttattgt ctggtgtttt 1140tattttcccc tgatagtcta atatgataaa ctctgcgttg tgaaaggtgg tggagcttga 1200ctttttgtac ccaagcgatg ggatacatag gaggtgggag aatgggtata gaataacatc 1260aatggcagca actgcggatc aagcagcttt catattaagc ataccaaagc gtaagatggt 1320ggatgaaact caagagactc tccgcaccac cgcctttcca agtactcatg tcaaggttgg 1380tttctttagc tttgaacaca gatttggatc tttttgtttt gtttccatat acttaggacc 1440tgagagcttt tggttgattt ttttttcagg acaaatgggc gaagaatctg tacattgcat 1500caatatgcta tggcaggaca gtgtgctgat acacacttaa gcatcatgtg gaaagccaaa 1560gacaattgga gcgagactca gggtcgtcat aataccaatc aaagacgtaa aaccagacgc 1620aacctctttg gttgaatgta atgaaaggga tgtgtcttgg tatgtatgta cgaataacaa 1680aagagaagat ggaattagta gtagaaatat ttgggagctt tttaagccct tcaagtgtgc 1740tttttatctt attgatatca tccatttgcg ttgtttaatg cgtctctaga tatgttccta 1800tatctttctc agtgtctgat aagtgaaatg tgagaaaacc ataccaaacc aaaatattca 1860aatcttattt ttaataatgt tgaatcactc ggagttgcca ccttctgtgc caattgtgct 1920gaatctatca cactagaaaa aaacatttct tcaaggtaat gacttgtgga ctatgttctg 1980aattctcatt aagtttttat tttctgaagt ttaagttttt accttctgtt ttgaaatata 2040tcgttcataa gatgtcacgc caggacatga gctacacatc gcacatagca tgcagatcag 2100gacgatttgt cactcacttc aaacacctaa gagcttctct ctcacagcgc acacacatat 2160gcatgcaata tttacacgtg atcgccatgc aaatctccat tctcacctat aaattagagc 2220ctcggcttca ctctttactc aaaccaaaac tcatcactac agaacataca caagataatt 2280cgtcgaggat ccgcggccgt cgaatcaaca agtttgtaca aaaaagcagg ctgcggccgc 2340acaatggctg ccgcgttaca attacaaaca cacccttgct tccatggcac gtgccaactc 2400tcacctccgc cacgaccttc cgtttccttc ccttcttcct cccgctcgtt tccatctagc 2460agacgttccc tgtccgcgca tgtgaaggcg gcggcgtcgt ctttgtccac caccaccttg 2520caggaaggga tagcggagtt ttacgatgag tcgtcgggga tttgggaaga catatggggt 2580gaccatatgc accatggata ttacgagccg ggttccgata tttcgggttc agatcatcgt 2640gccgctcaga ttcgaatggt cgaagaatcg ctccgttttg ctggaatatc agaggaccca 2700gcaaacaggc ccaagagaat agttgatgtt gggtgtggga taggaggcag ttctaggtat 2760ctagcaagga aatatggggc aaaatgccaa ggcattactt tgagccctgt tcaagctgga 2820agagccaatg ctcttgctaa tgctcaagga ctagcagaac aggtttgttt tgaagttgca 2880gatgccttga accaaccatt ccctgatgac caatttgatc ttgtttggtc tatggaaagc 2940ggagaacaca tgcctgacaa acccaagttt gttaaagagc tggtgcgagt ggcagctcca 3000ggaggcacaa taatagtagt gacatggtgc catagggatc ttggtccatc tgaagagtct 3060ttgcagccat gggagcaaaa gcttttaaac agaatatgtg atgcttacta tttaccagag 3120tggtgttcta cttctgatta tgtcaaatta tttcagtccc tatctctcca ggatataaag 3180gcaggagact ggactgagaa tgtagcaccc ttttggccag cagtgatacg ttcagcattg 3240acatggaagg gcttcacatc gctgctacga agtggattaa aaacaataaa aggtgcactg 3300gtgatgccat tgatgatcga aggtttccag aaaggggtga taaagtttgc catcattgct 3360tgccggaagc cagctgagta gcctgcagga cccagctttc ttgtacaaag tggttgatgg 3420tcgagagtgt gtataccacg gtgatatgag tgtggttgtt gatgtatgtt agcttgggga 3480caagtttgta caaaaaagca ggctgcggcc gccagtgtga tggatatctg cagaattcgg 3540cttcgccctt cggccgcgca tgatggtgaa gaaattgtcg acctttctct tgtctgtttg 3600tcttttgtta aagaagctat gcttcgttct aataatctta ttgtccattt tgttgtgtta 3660tgacattttg gctgctccca tggcaggtcc gtcgcttctc ttccatttct tctcattttc 3720gattttgatt cttatttctt tccagtagct cctgctctgt gaatttctcc gctcacgata 3780gatctgctta tactccttac attcaacctt agatctggtc tcgattctct gtttctctgt 3840ttttttcttt tggtcgagaa tctgatgttt gtttatgttc tgtcaccatt aataataatg 3900aactctctca ttcatacaat gattagtttc tctcgtctac aaaacgatat gttgcatttt 3960cacttttctt ctttttttct aagatgattt gctttgacca atttgtttag atctttattt 4020tattttattt tctggtgggt tggtggaaat tgaaaaaaaa aaaaaacagc ataaattgtt 4080atttgttaat gtattcattt tttggctatt tgttctgggt aaaaatctgc ttctactatt 4140gaatctttcc tggatttttt actcctattg ggtttttata gtaaaaatac ataataaaag 4200gaaaacaaaa gttttataga ttctcttaaa ccccttacga taaaagttgg aatcaaaata 4260attcaggatc agatgctctt tgattgattc agatgcgatt acagttgcat ggcaaatttt 4320ctagatccgt cgtcacattt tattttctgt ttaaatatct aaatctgata tatgatgtcg 4380acaaattctg gtggcttata catcacttca actgttttct tttggctttg tttgtcaact 4440tggttttcaa tacgatttgt gatttcgatc gctgaatttt taatacaagc aaactgatgt 4500taaccacaag caagagatgt gacctgcctt attaacatcg tattacttac tactagtcgt 4560attctcaacg caatcgtttt tgtatttctc acattatgcc gcttctctac tctttattcc 4620ttttggtcca cgcattttct atttgtggca atccctttca caacctgatt tcccactttg 4680gatcatttgt ctgaagactc tcttgaatcg ttaccacttg tttcttgtgc atgctctgtt 4740ttttagaatt aatgataaaa ctattccata gtcttgagtt ttcagcttgt tgattctttt 4800gcttttggtt ttctgcaggg taccgagcag ccaaaatgtc aaaacacaac aaaatggaca 4860ataagattat taaaacgaag catagcttct ttaacaaaag acaaacagac aagagaaagg 4920tcgacaattt cttcaccatc atgccccggg acccagcttt cttgtacaaa gtggtcccca 4980agctaacact acatagtcat ggtgtgtgtt ccataaataa tgtactaatg taataagaac 5040tactccgtag acggtaataa aagagaagtt ttttttttta ctcttgctac tttcctataa 5100agtgatgatt aacaacagat acaccaaaaa gaaaacaatt aatctatatt cacaatgaag 5160cagtactagt ctattgaaca tgtcagattt tctttttcta aatgtctaat taagccttca 5220aggctagtga tgataaaaga tcatccaatg ggatccaaca aagactcaaa tctggttttg 5280atcagatact tcaaaactat ttttgtattc attaaattat gcaagtgttc ttttatttgg 5340tgaagactct ttagaagcaa agaacgacaa gcagtaataa aaaaaacaaa gttcagtttt 5400aagatttgtt attgacttat tgtcatttga aaaatatagt atgatattaa tatagtttta 5460tttatataat gcttgtctat tcaagatttg agaacattaa tatgatactg tccacatatc 5520caatatatta agtttcattt ctgttcaaac atatgataag atggtcaaat gattatgagt 5580tttgttattt acctgaagaa aagataagtg agcttcgagt ttctgaaggg tacgtgatct 5640tcatttcttg gctaaaagcg aatatgacat cacctagaga aagccgataa tagtaaactc 5700tgttcttggt ttttggttta atcaaaccga accggtagct gagtgtcaag tcagcaaaca 5760tcgcaaacca tatgtcaatt cgttagattc ccggtttaag ttgtaaaccg gtatttcatt 5820tggtgaaaac cctagaagcc agccaccctt tttaatctaa tttttgtaaa cgagaagtca 5880ccacacctct ccactaaaac cctgaacctt actgagagaa gcagagcgca gctcaaagaa 5940caaataaaac ccgaagatga gaccaccacg tggcggcggg agcttcaggg gacggggagg 6000aagagatggc ggcggacgct ttggtggcgg cggcggacgt tttggtggcg gcggtggacg 6060ttttggtggc ggcggtggac gctttggtgg tggatatcgt gacgaaggac ctcccagtga 6120agtcattggt tcgtttactc ttttcttagt cgaatcttat tcttgctctg ctcgttgttt 6180taccgataaa gctaggtaca gcttggcact ggccgtcgtt ttacaacgtc gtgactggga 6240aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 6300taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 6360atggcgccaa gctcctcgag ctatctgtca cttcatcaaa aggacagtag aaaaggaagg 6420tggcacctac aaatgccatc attgcgataa aggaaaggct atcgttcaag atgcctctgc 6480cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 6540tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga 6600cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 6660ggagaggaca cgctgaaatc accagtctct ctctacaaat ctatctctct ctattttctc 6720cataataatg tgtgagtagt tcccagataa gggaattagg gttcttatag ggtttcgctc 6780atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 6840gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 6900caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 6960gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 7020aacgcctacg actggacggc cgagtcaacc gtgtacgtct ccccccgcca ccagcggacg 7080ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 7140agcgtggttg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 7200ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 7260gtgggtttct ggcagctgga cttcagcctg ccagtaccgc cccgtccggt cctgcccgtc 7320accgagattt gagaattgat cgttcaaaca tttggcaata aagtttctta agattgaatc 7380ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa 7440taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc 7500aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat 7560cgcgcgcggt gtcatctatg ttactagatc ctcgagcgat cgtgaagttt ctcatctaag 7620cccccatttg gacgtgaatg tagacacgtc gaaataaaga tttccgaatt agaataattt 7680gtttattgct ttcgcctata aatacgacgg atcgtaattt gtcgttttat caaaatgtac 7740tttcatttta taataacgct gcggacatct acatttttga attgaaaaaa aattggtaat 7800tactctttct ttttctccat attgaccatc atactcattg ctgatccatg tagatttccc 7860ggacatgaag ccatttacaa ttgaatatat cctgccgccg ctgccgcttt gcacccggtg 7920gagcttgcat gttggtttct acgcagaact gagccggtta ggcagataat ttccattgag 7980aactgagcca tgtgcacctt ccccccaaca cggtgagcga cggggcaacg gagtgatcca 8040catgggactt ttaaacatca tccgtcggat ggcgttgcga gagaagcagt cgatccgtga 8100gatcagccga cgcaccgggc aggcgcgcaa cacgatcgca aagtatttga acgcaggtac 8160aatcgagccg acgttcacg 8179 6 7713 DNA Artificial Sequence Vector 6cgaagctcgg tcccgtgggt gttctgtcgt ctcgttgtac aacgaaatcc attcccattc 60cgcgctcaag atggcttccc ctcggcagtt catcagggct aaatcaatct agccgacttg 120tccggtgaaa tgggctgcac tccaacagaa acaatcaaac aaacatacac agcgacttat 180tcacacgagc tcaaattaca acggtatata tcctgccagt cagcatcatc acaccaaaag 240ttaggcccga atagtttgaa attagaaagc tcgcaattga ggtctgcgcc caatacgcaa 300accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 360ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 420ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 480atttcacaca ggaaacagct atgaccatga ttacgaattg taccgaatta tcactacaat 540gtcggagaga caaggctgcg ccagcatata caaaagggaa atgaagatgg ccttttgatt 600agctgtgtag catcagcagc taatctctgg gctctcatca tggatgctgg aactggattc 660acttctcaag tttatgagtt gtcaccggtc ttcctacaca aggtaataat cagttgaagc 720aattaagaat caatttgatt tgtagtaaac taagaagaac ttaccttatg ttttccccgc 780aggactggat tatggaacaa tgggaaaaga actactatat aagctccata gctggttcag 840ataacgggag ctctttagtt gttatgtcaa aaggttagtg tttagtgaat aataaactta 900taccacaaag tcttcattga cttatttata tacttgttgt gaattgctag gaactactta 960ttctcagcag tcatacaaag tgagtgactc atttccgttc aagtggataa ataagaaatg 1020gaaagaagat tttcatgtaa cctccatgac aactgctggt aatcgttggg gtgtggtaat 1080gtcgaggaac tctggcttct ctgatcaggt aggtttttgt ctcttattgt ctggtgtttt 1140tattttcccc tgatagtcta atatgataaa ctctgcgttg tgaaaggtgg tggagcttga 1200ctttttgtac ccaagcgatg ggatacatag gaggtgggag aatgggtata gaataacatc 1260aatggcagca actgcggatc aagcagcttt catattaagc ataccaaagc gtaagatggt 1320ggatgaaact caagagactc tccgcaccac cgcctttcca agtactcatg tcaaggttgg 1380tttctttagc tttgaacaca gatttggatc tttttgtttt gtttccatat acttaggacc 1440tgagagcttt tggttgattt ttttttcagg acaaatgggc gaagaatctg tacattgcat 1500caatatgcta tggcaggaca gtgtgctgat acacacttaa gcatcatgtg gaaagccaaa 1560gacaattgga gcgagactca gggtcgtcat aataccaatc aaagacgtaa aaccagacgc 1620aacctctttg gttgaatgta atgaaaggga tgtgtcttgg tatgtatgta cgaataacaa 1680aagagaagat ggaattagta gtagaaatat ttgggagctt tttaagccct tcaagtgtgc 1740tttttatctt attgatatca tccatttgcg ttgtttaatg cgtctctaga tatgttccta 1800tatctttctc agtgtctgat aagtgaaatg tgagaaaacc ataccaaacc aaaatattca 1860aatcttattt ttaataatgt tgaatcactc ggagttgcca ccttctgtgc caattgtgct 1920gaatctatca cactagaaaa aaacatttct tcaaggtaat gacttgtgga ctatgttctg 1980aattctcatt aagtttttat tttctgaagt ttaagttttt accttctgtt ttgaaatata 2040tcgttcataa gatgtcacgc caggacatga gctacacatc gcacatagca tgcagatcag 2100gacgatttgt cactcacttc aaacacctaa gagcttctct ctcacagcgc acacacatat 2160gcatgcaata tttacacgtg atcgccatgc aaatctccat tctcacctat aaattagagc 2220ctcggcttca ctctttactc aaaccaaaac tcatcactac agaacataca caagataatt 2280cgtcgaggat ccgcggccgt cgaatcaaca agtttgtaca aaaaagcagg ctgcggccgc 2340acaatggctg ccgcgttaca attacaaaca cacccttgct tccatggcac gtgccaactc 2400tcacctccgc cacgaccttc cgtttccttc ccttcttcct cccgctcgtt tccatctagc 2460agacgttccc tgtccgcgca tgtgaaggcg gcggcgtcgt ctttgtccac caccaccttg 2520caggaaggga tagcggagtt ttacgatgag tcgtcgggga tttgggaaga catatggggt 2580gaccatatgc accatggata ttacgagccg ggttccgata tttcgggttc agatcatcgt 2640gccgctcaga ttcgaatggt cgaagaatcg ctccgttttg ctggaatatc agaggaccca 2700gcaaacaggc ccaagagaat agttgatgtt gggtgtggga taggaggcag ttctaggtat 2760ctagcaagga aatatggggc aaaatgccaa ggcattactt tgagccctgt tcaagctgga 2820agagccaatg ctcttgctaa tgctcaagga ctagcagaac aggtttgttt tgaagttgca 2880gatgccttga accaaccatt ccctgatgac caatttgatc ttgtttggtc tatggaaagc 2940ggagaacaca tgcctgacaa acccaagttt gttaaagagc tggtgcgagt ggcagctcca 3000ggaggcacaa taatagtagt gacatggtgc catagggatc ttggtccatc tgaagagtct 3060ttgcagccat gggagcaaaa gcttttaaac agaatatgtg atgcttacta tttaccagag 3120tggtgttcta cttctgatta tgtcaaatta tttcagtccc tatctctcca ggatataaag 3180gcaggagact ggactgagaa tgtagcaccc ttttggccag cagtgatacg ttcagcattg 3240acatggaagg gcttcacatc gctgctacga agtggattaa aaacaataaa aggtgcactg 3300gtgatgccat tgatgatcga aggtttccag aaaggggtga taaagtttgc catcattgct 3360tgccggaagc cagctgagta gcctgcaggc cgtcgcttct cttccatttc ttctcatttt 3420cgattttgat tcttatttct ttccagtagc tcctgctctg tgaatttctc cgctcacgat 3480agatctgctt atactcctta cattcaacct tagatctggt ctcgattctc tgtttctctg 3540tttttttctt ttggtcgaga atctgatgtt tgtttatgtt ctgtcaccat taataataat 3600gaactctctc attcatacaa tgattagttt ctctcgtcta caaaacgata tgttgcattt 3660tcacttttct tctttttttc taagatgatt tgctttgacc aatttgttta gatctttatt 3720ttattttatt ttctggtggg ttggtggaaa ttgaaaaaaa aaaaaacagc ataaattgtt 3780atttgttaat gtattcattt tttggctatt tgttctgggt aaaaatctgc ttctactatt 3840gaatctttcc tggatttttt actcctattg ggtttttata gtaaaaatac ataataaaag 3900gaaaacaaaa gttttataga ttctcttaaa ccccttacga taaaagttgg aatcaaaata 3960attcaggatc agatgctctt tgattgattc agatgcgatt acagttgcag ggcaaatttt 4020ctagatccgt cgtcacattt tatcttctgt ttaaatatct aaatctgata tatgatgtcg 4080acaaattctg gtggcttata catcacttca actgttttct tttggctttg tttgtcaact 4140tggttttcaa tacgatctgt gatttcgatc gctgaatttt taatacaagc aaactgatgt 4200taaccacaag caagagatgt gacctgcctt attaacatcg tattacttac tgctagtcgt 4260attctcaacg caatcgtttt tgtatttctc acattatgcc gcttctctac tctttattcc 4320ttttggtcca cgcattttct atttgtggca atccctttca caacctgatt tcccactttg 4380gatcatttgt ctgaagactc tcttgaatcg ttaccacttg tttcttgtgc atgctctgtt 4440ttttagaatt aatgataaaa ctattccata gtcttgagtt ttcagcttgt tgattctttt 4500gcttttggtt ttctgcccaa cactacatag tcatggtgtg tgttccataa ataatgtact 4560aatgtaataa gaactactcc gtagacggta ataaaagaga agtttttttt tttactcttg 4620ctactttcct ataaagtgat gattaacaac agatacacca aaaagaaaac aattaatcta 4680tattcacaat gaagcagtac tagtctattg aacatgtcag attttctttt tctaaatgtc 4740taattaagcc ttcaaggcta gtgatgataa aagatcatcc aatgggatcc aacaaagact 4800caaatctggt tttgatcaga tacttcaaaa ctatttttgt attcattaaa ttatgcaagt 4860gttcttttat ttggtgaaga ctctttagaa gcaaagaacg acaagcagta ataaaaaaaa 4920caaagttcag ttttaagatt tgttattgac ttattgtcat ttgaaaaata tagtatgata 4980ttaatatagt tttatttata taatgcttgt ctattcaaga tttgagaaca ttaatatgat 5040actgtccaca tatccaatat attaagtttc atttctgttc aaacatatga taagatggtc 5100aaatgattat gagttttgtt atttacctga agaaaagata agtgagcttc gagtttctga 5160agggtacgtg atcttcattt cttggctaaa agcgaatatg acatcaccta gagaaagccg 5220ataatagtaa actctgttct tggtttttgg tttaatcaaa ccgaaccggt agctgagtgt 5280caagtcagca aacatcgcaa accatatgtc aattcgttag attcccggtt taagttgtaa 5340accggtattt catttggtga aaaccctaga agccagccac cctttttaat ctaatttttg 5400taaacgagaa gtcaccacac ctctccacta aaaccctgaa ccttactgag agaagcagag 5460cgcagctcaa agaacaaata aaacccgaag atgagaccac cacgtggcgg cgggagcttc 5520aggggacggg gaggaagaga tggcggcgga cgctttggtg gcggcggcgg acgttttggt 5580ggcggcggtg gacgttttgg tggcggcggt ggacgctttg gtggtggata tcgtgacgaa 5640ggacctccca gtgaagtcat tggttcgttt actcttttct tagtcgaatc ttattcttgc 5700tctgctcgtt gttttaccga taaagctagg tacagcttgg cactggccgt cgttttacaa 5760cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 5820ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 5880agcctgaatg gcgaatggcg ccaagctcct cgagctatct gtcacttcat caaaaggaca 5940gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt 6000caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 6060gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact 6120gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga 6180agttcatttc atttggagag gacacgctga aatcaccagt ctctctctac aaatctatct 6240ctctctattt tctccataat aatgtgtgag tagttcccag ataagggaat tagggttctt 6300atagggtttc gctcatgagc ccagaacgac gcccggccga catccgccgt gccaccgagg 6360cggacatgcc ggcggtctgc accatcgtca accactacat cgagacaagc acggtcaact 6420tccgtaccga gccgcaggaa ccgcaggagt ggacggacga cctcgtccgt ctgcgggagc 6480gctatccctg gctcgtcgcc gaggtggacg gcgaggtcgc cggcatcgcc tacgcgggcc 6540cctggaaggc acgcaacgcc tacgactgga cggccgagtc aaccgtgtac gtctcccccc 6600gccaccagcg gacgggactg ggctccacgc tctacaccca cctgctgaag tccctggagg 6660cacagggctt caagagcgtg gttgctgtca tcgggctgcc caacgacccg agcgtgcgca 6720tgcacgaggc gctcggatat gccccccgcg gcatgctgcg ggcggccggc ttcaagcacg 6780ggaactggca tgacgtgggt ttctggcagc tggacttcag cctgccagta ccgccccgtc 6840cggtcctgcc cgtcaccgag atttgagaat tgatcgttca aacatttggc aataaagttt 6900cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta 6960cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat 7020gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa 7080ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcctcgag cgatcgtgaa 7140gtttctcatc taagccccca tttggacgtg aatgtagaca cgtcgaaata aagatttccg 7200aattagaata atttgtttat tgctttcgcc tataaatacg acggatcgta atttgtcgtt 7260ttatcaaaat gtactttcat tttataataa cgctgcggac atctacattt ttgaattgaa 7320aaaaaattgg taattactct ttctttttct ccatattgac catcatactc attgctgatc 7380catgtagatt tcccggacat gaagccattt acaattgaat atatcctgcc gccgctgccg 7440ctttgcaccc ggtggagctt gcatgttggt ttctacgcag aactgagccg gttaggcaga 7500taatttccat tgagaactga gccatgtgca ccttcccccc aacacggtga gcgacggggc 7560aacggagtga tccacatggg acttttaaac atcatccgtc ggatggcgtt gcgagagaag 7620cagtcgatcc gtgagatcag ccgacgcacc gggcaggcgc gcaacacgat cgcaaagtat 7680ttgaacgcag gtacaatcga gccgacgttc acg 7713

What is claimed is:
 1. A nucleic acid molecule comprising a firstnucleic acid segment comprising a polypeptide encoding sequence and asecond nucleic acid segment comprising a gene suppression sequence,wherein transcription of said nucleic acid molecule in a host cellresults in expression of a polypeptide by said polypeptide encodingsequence and suppression of a gene in said host cell.
 2. The nucleicacid molecule according to claim 1, wherein said second nucleic acidsegment is expressed as a dsRNA molecule.
 3. The nucleic acid moleculeaccording to claim 2, wherein said second nucleic acid segment has atleast 21 contiguous nucleotides corresponding to an mRNA.
 4. The nucleicacid molecule according to claim 3, wherein said second nucleic acidsegment has at least 21 contiguous nucleotides corresponding to anintron from said mRNA.
 5. The nucleic acid molecule according to claim3, wherein said second nucleic acid segment has at least 21 contiguousnucleotides corresponding to an exon from said mRNA.
 6. The nucleic acidmolecule according to claim 3, wherein said second nucleic acid segmenthas at least 21 contiguous nucleotides corresponding to a 3′ UTR fromsaid mRNA.
 7. The nucleic acid molecule according to claim 3, whereinsaid second nucleic segment acid has at least 21 contiguous nucleotidescorresponding to a 5′ UTR from said mRNA.
 8. The nucleic acid moleculeaccording to claim 1, wherein said first nucleic acid segment and saidsecond nucleic acid segment are operably linked to a single promoter. 9.The nucleic acid molecule according to claim 1, wherein said suppressionof a gene is suppression of an endogenous gene to said host cell.
 10. Aplant having in its genome a nucleic acid molecule of claim
 1. 11. Amethod of simultaneously altering the expression of more than one RNAmolecule in a plant comprising introducing into the genome of said planta nucleic acid molecule of claim
 1. 12. The method according to claim11, wherein said second nucleic acid segment is expressed as a dsRNAmolecule.
 13. The method according to claim 12, wherein said secondnucleic acid segment has at least 21 contiguous nucleotidescorresponding to an mRNA.
 14. The method according to claim 12, whereinsaid second nucleic acid segment has at least 21 contiguous nucleotidescorresponding to an intron from said mRNA.
 15. The method according toclaim 12, wherein said second nucleic acid segment has at least 21contiguous nucleotides corresponding to an exon from said mRNA.
 16. Themethod according to claim 12, wherein said second nucleic acid segmenthas at least 21 contiguous nucleotides corresponding to a 3′ UTR fromsaid mRNA.
 17. The method according to claim 12, wherein said secondnucleic acid segment has at least 21 contiguous nucleotidescorresponding to a 5′ UTR from said mRNA.
 18. The method according toclaim 11, wherein the level of expression of at least one of said morethan one RNA molecules is at least partially reduced.
 19. The methodaccording to claim 18, wherein said level of expression of at least oneof said more than one RNA molecules is substantially reduced.
 20. Themethod according to claim 19, wherein the level of expression of atleast one of said more than one RNA molecules is effectively eliminated.